Compounds and methods for treating aids and hiv infections

ABSTRACT

Macrocycle containing carbamate compounds that inhibit HIV proteolytic enzymes and processes for preparing them are described. Compositions and methods for treating a patient infected with HIV are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/319,973, filed Nov. 10, 2011, which is a national stage entry under35 U.S.C. §371(b) of International Application No. PCT/US2010/034437,filed May 11, 2010, which claims priority under 35 USC §119(e) to U.S.Provisional Application Ser. No. 61/177,086, filed on May 11, 2009, theentire disclosures of which are incorporated herein by reference

GOVERNMENT RIGHTS

This invention was made with government support under grant numberGM053386 awarded by the National Institutes of Health. The governmenthas certain rights in the invention,

TECHNICAL FIELD

This invention relates to compounds that inhibit HIV proteolytic enzymesand processes for preparing the compounds. The invention also relates tomethods of using the disclosed compounds for treating patients infectedwith HIV.

BACKGROUND AND SUMMARY

The AIDS epidemic is one of the most challenging problems in medicine inthe 21st century (United Nations. 2004 Report on the global HIV/AIDSEpidemic: 4th global report. New York, U.S.A., 2004). The disclosure ofthe foregoing is incorporated herein in its entirety by reference. Inaddition, the entirety of the disclosures of each of the publicationscited herein are also incorporated herein by reference. A retrovirusdesignated human immunodeficiency virus (HIV) is the etiological agentof the complex disease that includes progressive destruction of theimmune system (acquired immune deficiency syndrome; AIDS) anddegeneration of the central and peripheral nervous system. This viruswas previously known as LAV, HTLV-III, or ARV, A common feature ofretro-virus replication is the extensive post-translational processingof precursor polyproteins by a virally encoded protease to generatemature viral proteins required for virus assembly and function.Inhibition of this processing prevents the production of normallyinfectious virus. For example, Kohl (1988) demonstrated that geneticinactivation of the HIV encoded protease resulted in the production ofimmature, non-infectious virus particles. These results indicate thatinhibition of the HIV protease represents a viable method for thetreatment of AIDS and the prevention or treatment of infection by HIV.

The introduction of protease inhibitors (PIs) into highly activeantiretroviral therapy (HAART), a combination therapy based onco-administration of PIs with reverse-transcriptase inhibitors, markedthe beginning of a new era in HIV/AIDS chemotherapy. HAART treatmentregimens have led to a significant decline in the number of deaths dueto HIV infection in the developed World (Sepkowitz, 2001). Thedisclosure of the foregoing is incorporated herein in its entirety byreference. In addition, the entirety of the disclosure of each of thepublications cited herein is also incorporated herein by reference.Unfortunately there are a number of factors that severely limit currentHAART treatment regimens. High frequency of dosing, heavy pill burdenand issues of tolerability and toxicity can lead to poor adherence totreatment (Waters, 2007). The need for more potent, less toxic drugregimens is quite apparent.

Currently available combination chemotherapy typically using two reversetranscriptase inhibitors (RTIs) and boosted protease inhibitors (PIs) oran integrase inhibitor or highly active antiretroviral therapy (HAART)for human immunodeficiency virus type 1 (HIV-1) infection and AIDS hasbeen shown to suppress the replication of HIV-1 and significantly extendthe life expectancy of HIV-1-infected individuals. Indeed, severalrecent analyses have revealed that mortality rates for HIV infectedpersons have become much closer to general mortality rates since theintroduction of HAART, and that first line HAART with boosted PI-basedregimens resulted in less resistance within and across drug classes.

However, the ability to provide effective long-term antiretroviraltherapy for HIV-1 infection has become a complex issue since those whoinitially achieved favorable viral suppression to undetectable levelshave experienced treatment failure. In addition, it is evident that evenwith these anti-HIV-1 drugs, only partial immunologic reconstitution isattained in patients with advanced HIV-1 infection and it is likely thatHIV-1 will eventually acquire resistance to virtually any antiviralagents. Thus, it appears that the development of potent anddrug-resistant-deferring antiviral agents will continue to be requiredfor successful long-term control of HIV-1 infection and AIDS.

It is the rapid emergence of drug resistance however, that is proving tobe a most formidable problem. Mutations causing drug resistance arethought to occur spontaneously, through the recombination of mixed viralpopulations, and also due to drug pressure, particularly whenadministered at sub-standard doses (Pillay, 2006; Grabar, 2000;Wainberg, 1998; Harrigan, 2005). A growing number of patients aredeveloping multi-drug-resistant HIV-1 variants (Hertogs, 2000; Yerly,1999). There is ample evidence that these viral strains can betransmitted. Thus, the development of antiretroviral agents able tomaintain potency against resistant HIV strains has become an urgentpriority.

The proteolytic enzyme, HIV-1 protease is essential for viral assemblyand maturation (Roberts, 1990; Meek, 1990; McQuade, 1990). As aconsequence, design of specific inhibitors for HIV-1 protease has becomethe subject of immense interest. In 1996, protease inhibitors (PIs) wereintroduced in combination with reverse transcriptase inhibitors tobecome highly active antiretroviral therapy (HAART) (Flexner, 1998;Cihlar, 2000). This treatment regimen significantly increased lifeexpectancy, improved quality of life and decreased mortality andmorbidity among HIV/AIDS patients. Despite these notable advances, theemergence of drug-resistant HIV-1 variants is severely limiting theefficacy of HAART treatment regimens. Therefore, development of newbroad spectrum antiretroviral drugs that produce minimal adverse effectsremains an important therapeutic objective for the treatment of HIV/AIDS(Wainberg, 2000; Hertogs, 2000).

Recently, structure-based design of inhibitors maximizing interactionswithin the active site protease back-bone were described, as was thedevelopment of nonpeptide inhibitors (1-2) that have shown picomolarenzyme affinity and exceptional antiviral activity against bothwild-type and drug-resistant HIV-1 strains (Ghosh, 1998; Koh, 2003;Ghosh, 2002; Surleraux, 2005; Yoshimura, 2002; Koh, 2003). The X-raycrystallographic studies revealed that backbone conformation of mutantprotease is minimally distorted compared to wild-type HIV-1 proteases.Without being bound by theory, it is believed herein that maximizing‘back-bone binding’ may be an important design strategy to combatdrug-resistance (Ghosh, 2008). Inhibitor 1 (Daraunavir, TMC-114) wasrecently approved by the FDA for the treatment of drug resistant HIVstrains (on Jun. 23, 2006, the FDA approved new HIV treatment forpatients who do not respond to existing drugs). More recently, it hasbeen approved for all HIV/AIDS patients including pediatric AIDSpatients (On Oct. 21, 2008, FDA granted traditional approval to Prezista(darunavir), co-administered with ritonavir and with otherantiretroviral agents, for the treatment of HIV-1 infection intreatment-experienced adult patients. In addition to the traditionalapproval, a new dosing regimen for treatment-naïve patients wasapproved).

-   -   1 (Darunavir, y=NH₂)        -   (K_(i)=16 pM; ID₅₀=1.6 nM)    -   2 (TMC-126, y=OMe)        -   (K_(i)=14 pM; ID₅₀=1.2 nM)

Described herein are novel PIs with functionalities capable ofinteracting with the protein backbone as well as the introduction offlexible macrocycles involving P1′-P2′-ligands.

It has been discovered that protease inhibitors (PIs) containingfunctionalities that interact with the amino acid backbones of thecatalytic site of HIV-1 protease along with a flexible macrocyclic groupinvolving P1′-P2′-ligands are potent inhibitors and also show highactivity in more relevant cell-based assays. In addition, it has beendiscovered herein the such compounds including a flexible macrocyclicgroup involving P1′-P2′-ligands for effective repacking of the alteredPI-binding cavity of protease that emerges upon side chain mutations inPI-resistant HIV-1 variants are potent inhibitors of such otherwiseresistant variants. Without being bound by theory, it is believed hereinthat the high activity of the compounds described herein may be due tothe dual mode of action in both inhibiting the proteolytic activity ofthe protease, as well as in inhibiting the dimerization of the protease.

In one illustrative embodiment of the invention, a compound having theformula

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof isdescribed, wherein

R² and R³ are in each instance independently selected from the groupconsisting of hydrogen and a prodrug forming group;

R⁵ is alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, eachof which is optionally substituted;

X⁴ is carbonyl, S(O) or SO₂;

X⁵ is a bond, oxygen, unsubstituted nitrogen, substituted nitrogen,sulfur, S(O) or sulfone;

L¹ and L² are independently selected in each instance from the groupconsisting of alkylene, cycloalkylene, unsaturated alkylene,heteroalkylene, cycloheteroalkylene, and unsaturated heteroalkylene,each of which is optionally substituted;

W is a bond, (H)C═C(H), oxygen, sulfur, S(O), SO₂, C(O), or optionallysubstituted nitrogen;

Q² is a divalent carbocyle, heterocycle, aryl, or heteroaryl, each ofwhich is optionally substituted with one or more substituents; and

Z is selected from the group consisting of monocyclic heterocycle,bicyclic heterocycle and tricyclic heterocycle, each of which isoptionally substituted.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ and L² are independently selected in each instance from thegroup consisting of alkylene, unsaturated alkylene, heteroalkylene, andunsaturated heteroalkylene, each of which is optionally substituted isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein at least one of L¹ or L² is cycloalkylene or cycloheteroalkyleneis described.

It is appreciated that the compounds described herein may be used aloneor in combination with other compounds useful for treating suchdiseases, including those compounds that may operate by the same ordifferent modes of action. Further, it is appreciated that the compoundsand compositions described herein may be administered alone or withother compounds and compositions, such as other antiviral agents,immunomodulators, antibiotics, vaccines, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. GRL-216 (14c) and GRL-286 (14b) potently block the dimerizationof HIV-1 protease. To examine whether GRL-216 and GRL-286 exertedinhibition of HIV-1 protease dimerization, the FRET-based HIV-1expression system was employed (Koh, 2007). COST cells were transfectedwith pHIV-PR_(WT CFP) and pHIV-PR_(WT YFP) and exposed to variousconcentrations of either of the drugs and the CFP_(A/B) 551 ratios weredetermined at the end of 72-hr culture. The average CFP_(A/B) 552 ratioswere all less than 1.0 in the presence of >0.1 μM GRL-216 and GRL-286,indicating that both compounds effectively blocked HIV-1 proteasedimerization.

FIG. 2. In vitro selection of HIV-1 variants resistant to GRL-216, -246(15c), -286 and -396. HIV-1_(NL4-3) was propagated in MT-4 cells in thepresence of increasing concentrations of APV (), GRL-216 (▴), -246 (▪),-286 (□) or -396 (14d)(∘). Each passage of HIV-1 was conducted in acell-free fashion. APV was employed as a reference compound.

FIG. 3. GRL-216 (14c) fails to block protease dimerization withV82I/I84V substitutions. To examine whether an amino acidsubstitution(s) that emerged upon GRL-216 selection of HIV-1_(NL4-3),recombinant clones containing one of the following mutations: L24I,V82I, I84V, V82I/I84V, and L10I/L24I/M46L/L63P/V82I/I84V were generatedin the setting of the FRET-based HIV-1 expression assay (Koh, 2007).With L24I, V82I, or I84V alone, the activity of 0.1 μM GRL-216 to blockprotease dimerization was not affected; however, with either set ofV82I/I84V or L10I/L24I/M46L/L63P/V82I/I84V substitutions, GRL-216 failedto block the dimerization.

DETAILED DESCRIPTION

In one embodiment of the invention, a compound having the formula

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof isdescribed wherein

R² and R³ are in each instance independently selected from the groupconsisting of hydrogen and a prodrug forming group;

R⁵ is alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, eachof which is optionally substituted;

X⁴ is carbonyl, S(O) or SO₂;

X⁵ is a bond, oxygen, unsubstituted nitrogen, substituted nitrogen,sulfur, S(O) or sulfone;

L¹ and L² are independently selected in each instance from the groupconsisting of alkylene, cycloalkylene, unsaturated alkylene,heteroalkylene, cycloheteroalkylene, and unsaturated heteroalkylene,each of which is optionally substituted;

W is a bond, (H)C═C(H), oxygen, sulfur, S(O), SO₂, C(O), or optionallysubstituted nitrogen;

Q² is a divalent carbocyle, heterocycle, aryl, or heteroaryl, each ofwhich is optionally substituted with one or more substituents; and

Z is selected from the group consisting of monocyclic heterocycle,bicyclic heterocycle and tricyclic heterocycle, each of which isoptionally substituted.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ and L² are independently selected in each instance from thegroup consisting of alkylene, unsaturated alkylene, heteroalkylene, andunsaturated heteroalkylene, each of which is optionally substituted isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ is CH₂(CH₂)_(m)CH₂; and m is from 0 to about 4 is described.

In another embodiment, the compound of any of the preceding embodimentswherein L² is CH₂(CH₂)_(n)CH₂; and n is from 0 to about 4 is described.

In another embodiment, the compound of any of the preceding embodimentswherein W is a bond, (H)C═C(H), oxygen, sulfur, S(O), S(O)₂, oroptionally substituted nitrogen is described.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ is CH₂(CH₂)_(m)CH₂; L² is CH₂(CH₂)_(n)CH₂; W is a bond,(H)C═C(H), oxygen, sulfur, S(O), S(O)₂, or optionally substitutednitrogen; m is from 0 to about 4; and n is from 0 to about 4 isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ is CH₂CH₂CH₂CH₂ is described.

In another embodiment, the compound of any of the preceding embodimentswherein L¹ is CH₂ is described.

In another embodiment, the compound of any of the preceding embodimentswherein m is 2; n is 0; and W is Z—(H)C═C(H) is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁵ is oxygen; and W is a bond; and L¹ and L² are alkylene isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein W is a bond; L¹ is CH₂CH₂CH₂CH₂; and L² is CH₂CH₂CH₂ isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein R³ is hydrogen is described.

In another embodiment, the compound of any of the preceding embodimentswherein at least one of L¹ or L² is cycloalkylene or cycloheteroalkyleneis described.

In another embodiment, the compound of the preceding embodiment whereinL¹ is cycloheteroalkylene where the cyclic portion is a pyrrolidinone isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein one of L¹ or L² is

where r is 1 or 2

In another embodiment, the compound of any of the preceding embodimentswherein one of L¹ or L² is

where r is 1 or 2

In another embodiment, the compound of any of the preceding embodimentswherein L¹ is

where r is 1, 2, 3, or 4 is described.

In another embodiment, the compound of any of the preceding embodimentswherein one of L¹ or L² is

where r is 1 or 2; and R^(A) is a carbonyl oxygen, hydroxyl, alkoxyl,amino, or heteroarylalkylamino is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁵ is oxygen is described.

In another embodiment, the compound of any of the preceding embodimentswherein W is (H)C═C(H) is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁵ is oxygen; and W is (H)C═C(H) is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁴ is S(O)₂ is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁴ is SO₂; and X⁵ is oxygen

In another embodiment, the compound of any of the preceding embodimentswherein Y² is methoxy is described.

In another embodiment, the compound of any of the preceding embodimentswherein Z is a bicycle heterocycle comprising at least one oxygen isdescribed.

In another embodiment, the compound of any of the preceding embodimentswherein Z has the formula,

each of which is optionally substituted, wherein

* indicates the point of attachment; s is 0 to 2; t is 0 to 4;

W¹ is optionally substituted alkylene or optionally substitutednitrogen;

W² represents between 1 and 11 divalent radicals each independentlyselected from the group consisting of optionally substituted alkylene,oxygen, sulfur, optionally substituted nitrogen, and SO₂; and

W³ is optionally substituted alkylene or oxygen is described.

In another embodiment, the compound of any of the preceding embodimentswherein Z has formula

where * indicates the point of attachment is described.

In another embodiment, the compound of any of the preceding embodimentswherein R⁵ is optionally substituted arylalkyl is described.

In another embodiment, the compound of any of the preceding embodimentswherein R⁵ is optionally substituted benzyl is described.

In another embodiment, the compound of any of the preceding embodimentswherein R³ is hydrogen is described.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is optionally substituted with one or more substituents Y²;wherein each substituent Y² is independently selected in each instancefrom the group consisting of hydroxy, halo, alkoxy, C(O)R⁸, C(O)NR⁹R¹⁰,C(O)OR⁸, SR⁸, S(O)R⁷, S(O)₂R⁷, NR⁹R¹⁰, alkylene-NR⁹R¹⁰, or alkyl,heteroalkyl, haloalkyl, cycloalkyl, alkenyl, alkylene-OR⁸, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each of which is optionallysubstituted;

where R⁷ is in each instance independently selected from the groupconsisting of alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, eachof which is optionally substituted; R⁸ is in each instance independentlyselected from the group consisting of alkyl, alkenyl, heteroalkyl,haloalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,and heteroarylalkyl, each of which is optionally substituted, or R⁸ ishydrogen; and R⁹ and R¹⁰ are in each instance independently selectedfrom hydrogen, or the group consisting of alkyl, alkenyl, heteroalkyl,haloalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,alkylsulfonyl, arylsulfonyl, and heteroarylalkyl, each of which isoptionally substituted; or R⁹ and R¹⁰ and the attached nitrogen form anoptionally substituted heterocycle is described.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is 1,2-phenylene, optionally substituted with one or more Y²is described.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is 1,2-phenylene substituted with at least one Y² isdescribed. In another embodiment, the compound of any of the precedingembodiments wherein X⁴ is SO₂; X⁵ is oxygen; and Q² is4-methoxy-1,2-phenylene is described.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is bicyclic is described.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is a heterocycle of the formula

optionally substituted with one or more substituents Y², where q is 1 or2 is described.

In another embodiment, a compound having the formula

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof isdescribed wherein

R² and R³ are in each instance independently selected from the groupconsisting of hydrogen and a prodrug forming group;

R⁵ is alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroaryl alkyl, eachof which is optionally substituted;

X⁴ is carbonyl, S(O) or SO₂;

X⁵ is a bond, oxygen, unsubstituted nitrogen, substituted nitrogen,sulfur, S(O) or sulfone;

L³ and L⁴ are independently selected in each instance from the groupconsisting of alkenyl, cycloalkenyl, heteroalkenyl, andcycloheteroalkenyl, each of which is optionally substituted, where bothL³ and L⁴ terminate in a carbon-carbon double bond;

Q² is divalent carbocyle, heterocycle, aryl, or heteroaryl, each ofwhich is optionally substituted with one or more substituents; and

Z is selected from the group consisting of monocyclic heterocycle,bicyclic heterocycle and tricyclic heterocycle, each of which isoptionally substituted.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is substituted with a least one Y² wherein each substituentY² is independently selected in each instance from the group consistingof hydroxy, halo, alkoxy, C(O)R⁸, C(O)NR⁹R¹⁰, C(O)OR⁸, SR⁸, S(O)R⁷,S(O)₂R⁷, NR⁹R¹⁰, alkylene-NR⁹R¹⁰, or alkyl, heteroalkyl, haloalkyl,cycloalkyl, alkenyl, alkylene-OR⁸, aryl, arylalkyl, heteroaryl andheteroarylalkyl, each of which is optionally substituted,

where R⁷ is in each instance independently selected from the groupconsisting of alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, eachof which is optionally substituted;

R⁸ is in each instance independently selected from the group consistingof alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted, or R⁸ is hydrogen; and

R⁹ and R¹⁰ are in each instance independently selected from hydrogen, orthe group consisting of alkyl, alkenyl, heteroalkyl, haloalkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,alkylsulfonyl, arylsulfonyl, and heteroarylalkyl, each of which isoptionally substituted; or R⁹ and R¹⁰ and the attached nitrogen form anoptionally substituted heterocycle;

In another embodiment, a pharmaceutical composition comprising thecompound of any of the preceding embodiments in a therapeuticallyeffective amount for treating HIV infection, and one or more of acarrier, diluent, excipient therefor, or a combination thereof isdescribed.

In another embodiment, a method for treating a patient in need of relieffrom HIV/AIDS disease, the method comprising the step of administeringto the patient a therapeutically effective amount of the composition ofany one of the preceding embodiments, or a composition comprising thecompound of any one of the preceding embodiments is described.

In another embodiment, the method or composition of any of the precedingembodiments wherein the HIV/AIDS disease includes at least one resistantHIV protease is described.

In another embodiment, the method or composition of any of the precedingembodiments wherein the HIV/AIDS disease is resistant to at least one ofsaquinavir, ritonavir, nelfinavir, lopinavir or atazanavir is described.

In another embodiment, the method or composition of any of the precedingembodiments wherein the HIV/AIDS disease includes at least onemulti-PI-resistant clinical HIV-1 variant is described.

In another embodiment, the compound having the formula

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof isdescribed; wherein

R² and R³ are in each instance independently selected from the groupconsisting of hydrogen and a prodrug forming group;

R⁵ is alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, eachof which is optionally substituted;

X⁴ is carbonyl, S(O) or SO₂;

X⁵ is a bond, oxygen, unsubstituted nitrogen, substituted nitrogen,sulfur, S(O) or sulfone;

L¹ and L² are independently selected in each instance from the groupconsisting of alkylene, unsaturated alkylene, heteroalkylene, andunsaturated heteroalkylene, each of which is optionally substituted;

W is a bond, (H)C═C(H), oxygen, sulfur, S(O), SO₂, C(O), or optionallysubstituted nitrogen;

Q² is divalent carbocyle, heterocycle, aryl, or heteroaryl, each ofwhich is optionally substituted with one or more substituents Y²;wherein each substituent Y² is independently selected in each instancefrom the group consisting of hydroxy, halo, alkoxy, C(O)R⁸, C(O)NR⁹R¹⁰,C(O)OR⁸, SR⁸, S(O)R⁷, S(O)₂R⁷, NR⁹R¹⁰, alkylene-NR⁹R¹⁰, or alkyl,heteroalkyl, haloalkyl, cycloalkyl, alkenyl, alkylene-OR⁸, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each of which is optionallysubstituted;

where R⁷ is in each instance independently selected from the groupconsisting of alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, eachof which is optionally substituted; R⁸ is in each instance independentlyselected from the group consisting of alkyl, alkenyl, heteroalkyl,haloalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,and heteroarylalkyl, each of which is optionally substituted, or R⁸ ishydrogen; and R⁹ and R¹⁰ are in each instance independently selectedfrom hydrogen, or the group consisting of alkyl, alkenyl, heteroalkyl,haloalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,alkylsulfonyl, arylsulfonyl, and heteroarylalkyl, each of which isoptionally substituted; or R⁹ and R¹⁰ and the attached nitrogen form anoptionally substituted heterocycle; and

Z is selected from the group consisting of monocyclic heterocycle,bicyclic heterocycle and tricyclic heterocycle, each of which isoptionally substituted.

In another embodiment, the compound of the preceding embodiment whereinL¹ is CH₂(CH₂)_(m)CH₂; L² is CH₂(CH₂)_(n)CH₂; W is a bond, (H)C═C(H),oxygen, sulfur, S(O), S(O)₂, or optionally substituted nitrogen; m isfrom 0 to about 4; and n is from 0 to about 4 is described. In anotherembodiment, the compound of the preceding embodiment wherein L¹ isCH₂CH₂CH₂CH₂ or CH₂ is described.

In another embodiment, the compound of any one of the precedingembodiments wherein X⁵ is oxygen; and W is (H)C═C(H) is described. Inanother embodiment, any of the preceding compounds wherein W isZ—(H)C═C(H) is described.

In another embodiment, the compound of any of the preceding embodimentswherein m is 2; n is 0; and W is Z—(H)C═C(H) is described.

In another embodiment, the compound of any of the preceding embodimentswherein X⁵ is oxygen; and W is a bond; and L¹ and L² are alkylene isdescribed. In another embodiment, the compound of any of the precedingembodiments wherein W is a bond; L¹ is CH₂CH₂CH₂CH₂; and L² is CH₂CH₂CH₂is described.

In another embodiment, the compound of formula

or a pharmaceutically acceptable salt, isomer, mixture of isomers,crystalline form, non crystalline form, hydrate, or solvate thereof isdescribed; wherein

R² and R³ are in each instance independently selected from the groupconsisting of hydrogen and a prodrug forming group;

R⁵ is alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, eachof which is optionally substituted;

X⁴ is carbonyl, S(O) or SO₂;

X⁵ is a bond, oxygen, unsubstituted nitrogen, substituted nitrogen,sulfur, S(O) or sulfone;

L³ and L⁴ are independently selected in each instance from the groupconsisting of unsaturated alkenyl, and heteroalkenyl, each of which isoptionally substituted;

Q² is divalent carbocyle, heterocycle, aryl, or heteroaryl, each ofwhich is optionally substituted with one or more substituents Y²;

wherein each substituent Y² is independently selected in each instancefrom the group consisting of hydroxy, halo, alkoxy, C(O)R⁸, C(O)NR⁹R¹⁰,C(O)OR⁸, SR⁸, S(O)R⁷, S(O)₂R⁷, NR⁹R¹⁰, alkylene-NR⁹R¹⁰, or alkyl,heteroalkyl, haloalkyl, cycloalkyl, alkenyl, alkylene-OR⁸, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each of which is optionallysubstituted,

where R⁷ is in each instance independently selected from the groupconsisting of alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, eachof which is optionally substituted;

R⁸ is in each instance independently selected from the group consistingof alkyl, alkenyl, heteroalkyl, haloalkyl, cycloalkyl, heterocycloalkyl,aryl, arylalkyl, heteroaryl, and heteroarylalkyl, each of which isoptionally substituted, or R⁸ is hydrogen; and

R⁹ and R¹⁰ are in each instance independently selected from hydrogen, orthe group consisting of alkyl, alkenyl, heteroalkyl, haloalkyl,cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl,alkylsulfonyl, arylsulfonyl, and heteroarylalkyl, each of which isoptionally substituted; or R⁹ and R¹⁰ and the attached nitrogen form anoptionally substituted heterocycle;

Z is selected from the group consisting of monocyclic heterocycle,bicyclic heterocycle and tricyclic heterocycle, each of which isoptionally substituted. In another embodiment, the compound of any ofthe preceding embodiments wherein Z is a bicycle heterocycle comprisingat least one oxygen is described.

In another embodiment, the compound of any of the preceding embodimentswherein Z has the formula,

each of which is optionally substituted is described, wherein

* indicates the point of attachment; s is 0 to 2; t is 0 to 4;

W¹ is optionally substituted alkylene or optionally substitutednitrogen;

W² represents between 1 and 11 divalent radicals each independentlyselected from the group consisting of optionally substituted alkylene,oxygen, sulfur, optionally substituted nitrogen, and SO₂; and

W³ is optionally substituted alkylene or oxygen.

In another embodiment, the compound of any of the preceding embodimentswherein Z has formula

where * indicates the point of attachment is described. In anotherembodiment, the compound of any of the preceding embodiments wherein Zhas the formula

where * indicates the point of attachment, is described. In anotherembodiment, the compound of the preceding embodiment wherein m is 2; nis 0; and W is Z—(H)C═C(H) is described.

The compound of any one of the preceding embodiments wherein R⁵ isoptionally substituted arylalkyl.

In another embodiment, the compound of any of the preceding embodimentswherein Q² is 1,2-phenylene, optionally substituted with one or more Y²,where Y² is as defined above is described. In another embodiment, thecompound of any of the preceding embodiments wherein X⁴ is SO₂; X⁵ isoxygen; Q² is 1,2-phenylene; and Y² is 4-methoxy is described. Inanother embodiment, the compound of any of the preceding embodimentswherein R⁵ is benzyl is described.

In another embodiment, also described herein are pharmaceuticalcompositions containing one or more of the compounds described herein.In one aspect, the compositions include a therapeutically effectiveamount of the one or more compounds for treating a patient withHIV/AIDS. It is to be understood that the compositions may include othercomponent and/or ingredients, including, but not limited to, othertherapeutically active compounds, and/or one or more carriers, diluents,excipients, and the like. In another embodiment, methods for using thecompounds and pharmaceutical compositions for treating patients withHIV/AIDS are also described herein. In one aspect, the methods includethe step of administering one or more of the compounds and/orcompositions described herein to a patient with HIV/AIDS. In anotheraspect, the methods include administering a therapeutically effectiveamount of the one or more compounds and/or compositions described hereinfor treating patients with HIV/AIDS. In another embodiment, uses of thecompounds and compositions in the manufacture of a medicament fortreating patients with HIV/AIDS are also described herein. In oneaspect, the medicaments include a therapeutically effective amount ofthe one or more compounds and/or compositions for treating a patientwith HIV/AIDS.

It is appreciated herein that the compounds described herein may be usedalone or in combination with other compounds useful for treatingHIV/AIDS, including those compounds that may be therapeuticallyeffective by the same or different modes of action. In addition, it isappreciated herein that the compounds described herein may be used incombination with other compounds that are administered to treat othersymptoms of HIV/AIDS, such as compounds administered to treat bacterial,fungal, and protozoic infections, and the like. In another embodiment, apharmaceutical composition comprising the compound of any one of thepreceding claims in a therapeutically effective amount for treatingHIV/AIDS disease, and one or more of a carrier, diluent, excipienttherefor, or a combination thereof is described.

In another embodiment, a method of treating a patient in need of relieffrom HIV infection, the method comprising the step of administering tothe patient a therapeutically effective amount of the composition of anyone of the preceding embodiments, or a composition comprising thecompound of any one of the preceding embodiments is described.

The synthesis of sulfonyl chlorides 7a-d is shown in Scheme 1. AMitsunobu-type reaction between 3-methoxyphenol and alcohols 4a-d in thepresence of triphenylphosphine and diisopropyl azodicarboxylate (DIAD)afforded ethers 5a-d. (Mitsunobu, 1981). Electrophilic aromaticsubstitution of ethers 5a-d with acetic anhydride and concentratedsulfuric acid in methanol furnished a mixture of sulfonic acidregioisomers 6a-d and 6e-h in a 1:1 ratio that were separated by flashchromatography. Structural confirmation of the isomers was determined byextensive 2D NMR experiments (NOESY and HMBC). Conversion to thesulfonyl chlorides 7a-d was achieved by reaction of the sulfonic acids6a-d with thionyl chloride in the presence of pyridine. It isappreciated that other compounds of the invention can be prepared usingmodifications well-known to the person having ordinary skill in the artof organic synthesis of schemes described herein.

In one embodiment, compounds 13a-h are synthesized as outlined in Scheme2. Nucleophilic attack of amines 9a and 9b on commercially availableepoxide 8 in the presence of isopropanol gave hydroxy amines 10a and10b. The conversion of amines 10a and 10b to the sulfonamides 11a-h wasrealized by coupling with sulfonyl chlorides 5a-d in the presence ofpyridine. Removal of the Boc protecting group from sulfonamides 11a-husing 30% trifluoroacetic acid in CH₂Cl₂ furnished the correspondingamines, which were then coupled with activated bis-THF (12) to giveacyclic inhibitors 13a-h (Ghosh, 2006).

In another embodiment, the acyclic compounds 13a-h thus obtained wereexposed to ring closing metathesis using Grubbs' 1st or 2nd generationcatalyst (Scheme 3) to give the unsaturated macrocyclic inhibitors 14a-h(Schwab, 1995; Scholl, 1999). Larger ring sizes (15-13) gave a mixtureof E/Z isomers, while in the case of smaller rings (12-9) the Z isomerwas obtained almost exclusively. The E and Z isomers were isolated usingreversed-phase HPLC and the stereochemistry established by 2-D NMR (COSYand NOESY) experiments allowing their individual biologicalcharacterization. In a further embodiment, the unsaturated compoundswere subsequently reduced using hydrogen and 10% Pd—C as a catalystyielding inhibitors 15a-g.

A series of methylated inhibitors were prepared in a similar fashion.Nucleophilic attack of amines 16a-c on commercially available epoxide 8gave hydroxy amines 17a-c (Scheme 4). The conversion of amines 17a-c tothe sulfonamides 18a-c was realized by coupling with sulfonyl chlorides7d in the presence of pyridine. Removal of the Boc protecting group fromsulfonamides 18a-c using 30% trifluoroacetic acid in CH₂Cl₂ furnishedthe corresponding amines, which were then coupled with activated bis-THF(12) to give acyclic inhibitors 19a-c (Ghosh, 2006). A ring closingmetathesis reaction using Grubbs' 2nd generation catalyst (Scheme 5)provided unsaturated macrocyclic inhibitors 20a-c and 21a-c, which wereseparated into their E/Z isomers by reversed-phase HPLC and identifiedby 2-D NMR (COSY and NOESY) (Schwab, 1999). The unsaturated compounds20a-c and 21a-c were subsequently reduced using hydrogen and 10% Pd—C asa catalyst yielding inhibitors 22a-c.

Inhibitors with longer carbon chains resulted in lower enzyme inhibitoryactivity. Extension of the β hydroxyl amine chain by three methylenegroups resulted in a 10 fold loss in activity (13a K_(i)=16 nM versus13e K_(i)=1.7 nM). Similarly, extension of the ether carbon chain bythree methylene groups resulted in a 17 fold loss in activity (13hK_(i)=0.10 nM versus 13e K_(i)=1.7 nM).

For example, the 14 membered macrocyclic inhibitors 14b and 15b haveK_(i) values less than 0.7 nM and IC₅₀ values less than 49 nM whereastheir corresponding acyclic inhibitor 13b had a K_(i) of 11 nM and IC₅₀value of >1000 nM (greater than 15-fold and 20-fold change,respectively). Without being bound by theory, Another trend observed isa preference of the S2′ subsite for macrocylic rings of size 10 and 13.Indeed, the most potent compound of the series, inhibitor 14cincorporating a 13 membered ring, showed a K_(i) of 45 μm and IC₅₀ of 2nm. Increasing the ring size to 14 or 15 as well as decreasing the ringto 12 or 11 resulted in reduced enzymatic inhibitory and antiviralactivity. Similarly, inhibitors incorporating a 10-membered ring (14gand 15f) were also very potent (14g K_(i)=51 μm; 15f K_(i)=86 μm andIC₅₀=5.5 nm).

The improvement in the K_(i) and IC₅₀ values for the unsaturated cyclicinhibitors 14a-h compared to their saturated analogs 15a-g is described.For the 13-membered ring series, the presence of a double bond resultsin a 10-fold increase in both K_(i) and IC₅₀ (14c K_(i)=45 pM and IC₅₀=2nM compared to 15c K_(i)=470 pM and IC₅₀=22 nM). Similar differences inpotency are observed for the 11, 14, and 15-membered macrocyclesalthough for the smaller rings 9, 10, and 12 the effect is lesspronounced. Without being bound by theory, it is believed that this mayresult from a restricted conformation in the molecule that results fromthe presence of the double bond and leads to a better fit in thehydrophobic pocket of the S2 subsite. The importance of thestereochemistry of the double bond in the olefinic compounds isdescribed. As shown in Table 3, only minor variations in activity (lessthan 5 fold) were observed between the E and Z isomers of the 13, 14,and 15-membered. For the 13-membered ring system the Z isomer wasfavored over the E configuration.

In another embodiment, possible substitutions that can be made acrossthe macrocylic ring system that further enhance biological activity aredescribed. Without being bound by theory, it is believed that a singlemethyl substitution β to the macrocylic amine atom could fill ahydrophobic pocket filled by a methyl group of darunavir's isopropylmoiety. The design and synthesis of a series of mono and dimethylated14-membered macrocylic ring systems and evaluation of the impact of thissubstitution on the biological activity is described. Geminal dimethylsat this location (19a through 22a) decreased (10-fold) enzyme inhibitionand reduced antiviral activity. Addition of a single methyl group to thering reduced biological activity as compared to 14b and 15b, althoughresults varied depending upon the stereochemistry of the ring systems.20c and 21b were the most potent compounds from this series withK_(i)=0.31 and 2.8 nM and IC₅₀=9.0 and 6.3 nM, respectively.

In another embodiment, compounds of formulae

-   -   m is 0-4; n is 0-4;    -   X=O, S, SO₂, NR, CHR, CR₂;    -   X′=O, S, SO₂, NR, CHR, CR₂;    -   Z=SO₂R, NR₂, CHROR, CR₃; where    -   where R is independently H, alkyl, heteroalkyl, alkylheterocycle

-   -   m is 0-4; n is 0-4;    -   X=O, S, SO₂, NR, CHR, CR₂;    -   X′=O, S, SO₂, NR, CHR, CR₂;    -   Z=SO₂R, NR₂, CHROR, CR₃, OR; where    -   where R is independently H, alkyl, heteroalkyl, alkylheterocycle        are described.

In another embodiment, compounds of formulae

are described.

In another embodiment, compounds of formulae

are described.

While certain embodiments of the present invention have been describedand/or exemplified above, it is contemplated that considerable variationand modification thereof are possible. Accordingly, the presentinvention is not limited to the particular embodiments described and/orexemplified herein.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers.Accordingly, it is to be understood that the present invention includespure stereoisomers as well as mixtures of stereoisomers, such asenantiomers, diastereomers, and enantiomerically or diastereomericallyenriched mixtures. The compounds described herein may be capable ofexisting as geometric isomers. Accordingly, it is to be understood thatthe present invention includes pure geometric isomers or mixtures ofgeometric isomers.

It is appreciated that the compounds described herein may exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms are equivalent to unsolvated forms and areencompassed within the scope of the present invention. The compounds ofthe present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

It is also appreciated that in the foregoing embodiments, certainaspects of the compounds are presented in the alternative, such asselections for any one or more of Z, Q², X⁴, X⁵, L¹, L², L³, L⁴, W, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, Y², n, m, s, and t. It is therefore tobe understood that various alternate embodiments of the inventioninclude individual members of those lists, as well as the varioussubsets of those lists. Each of those combinations is to be understoodto be described herein by way of the lists.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched. As used herein, the term “alkenyl” and “alkynyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond or triple bond, respectively. It is tobe understood that alkynyl may also include one or more double bonds. Itis to be further understood that alkyl is advantageously of limitedlength, including C₁-C₂₄, C₁-C₁₂, C₁-C₈, C₁-C₆, and C₁-C₄. It is to befurther understood that alkenyl and/or alkynyl may each beadvantageously of limited length, including C₂-C₂₄, C₂-C₁₂, C₂-C₈,C₂-C₆, and C₂-C₄. It is appreciated herein that shorter alkyl, alkenyl,and/or alkynyl groups may add less lipophilicity to the compound andaccordingly will have different pharmacokinetic behavior.

As used herein, the term “cycloalkyl” includes a chain of carbon atoms,which is optionally branched, where at least a portion of the chain incyclic. It is to be understood that cycloalkylalkyl is a subset ofcycloalkyl. It is to be understood that cycloalkyl may be polycyclic.Illustrative cycloalkyl include, but are not limited to, cyclopropyl,cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl,adamantyl, and the like. As used herein, the term “cycloalkenyl”includes a chain of carbon atoms, which is optionally branched, andincludes at least one double bond, where at least a portion of the chainin cyclic. It is to be understood that the one or more double bonds maybe in the cyclic portion of cycloalkenyl and/or the non-cyclic portionof cycloalkenyl. It is to be understood that cycloalkenylalkyl andcycloalkylalkenyl are each subsets of cycloalkenyl. It is to beunderstood that cycloalkyl may be polycyclic. Illustrative cycloalkenylinclude, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl,cycloheptenylpropenyl, and the like. It is to be further understood thatchain forming cycloalkyl and/or cycloalkenyl is advantageously oflimited length, including C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₅-C₆. It isappreciated herein that shorter alkyl and/or alkenyl chains formingcycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicityto the compound and accordingly will have different pharmacokineticbehavior.

As used herein, alkylene includes bivalent hydrocarbon groups whereinthe hydrocarbon group may be a straight-chained or a branched-chainhydrocarbon group. Non-limiting illustrative examples include methylene,1,2-ethylene, 1-methyl-1,2-ethylene, 1,4-butylene,2,3-dimethyl-1,4-butylene, 2-methyl-2-ethyl-1,5-pentylene, and the like.

As used herein, the term “cycloalkylene” includes a bivalent chain ofcarbon atoms, which is optionally branched, where at least a portion ofthe chain in cyclic. It is to be understood that alkylcycloalkylalkyl isa subset of cycloalkylene. It is to be understood that cycloalkylene maybe polycyclic.

As used herein, the term “heteroalkyl” includes a chain of atoms thatincludes both carbon and at least one heteroatom, and is optionallybranched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur.In certain variations, illustrative heteroatoms also include phosphorus,and selenium. As used herein, the term “cycloheteroalkyl” includingheterocyclyl and heterocycle, includes a chain of atoms that includesboth carbon and at least one heteroatom, such as heteroalkyl, and isoptionally branched, where at least a portion of the chain is cyclic.Illustrative heteroatoms include nitrogen, oxygen, and sulfur. Incertain variations, illustrative heteroatoms also include phosphorus,and selenium. Illustrative cycloheteroalkyl include, but are not limitedto, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

As used herein, the term “heteroalkylene” includes an alkylene groupwherein one or more carbon atoms are replaced with a hetero atomselected from oxygen, sulfur or optionally substituted nitrogen.

As used herein, the term “cycloheteroalkylene” includes a bivalent chainof atoms that includes both carbon and at least one heteroatom, such asheteroalkylene, and is optionally branched, where at least a portion ofthe chain is cyclic. Illustrative hetero atoms include nitrogen, oxygen,and sulfur. It is to be understood that heteroatoms may be included inthe cyclic portion, the non-cyclic portion, or in both the cyclic andnon-cyclic portions of the cycloheteroalkylene.

As used herein, the term “aryl” includes monocyclic and polycyclicaromatic groups, including aromatic carbocyclic and aromaticheterocyclic groups, each of which may be optionally substituted. Asused herein, the term “carbaryl” includes aromatic carbocyclic groups,each of which may be optionally substituted. Illustrative aromaticcarbocyclic groups described herein include, but are not limited to,phenyl, naphthyl, and the like. As used herein, the term “heteroaryl”includes aromatic heterocyclic groups, each of which may be optionallysubstituted. Illustrative aromatic heterocyclic groups include, but arenot limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl,imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl,thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl,benzisoxazolyl, benzisothiazolyl, and the like.

As used herein, the term “amino” includes the group NH₂, alkylamino, anddialkylamino, where the two alkyl groups in dialkylamino may be the sameor different, i.e. alkylalkylamino. Illustratively, amino includesmethylamino, ethylamino, dimethylamino, methylethylamino, and the like.In addition, it is to be understood that when amino modifies or ismodified by another term, such as aminoalkyl, or acylamino, the abovevariations of the term amino are included therein. Illustratively,aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl,dimethylaminoalkyl, methylethylaminoalkyl, and the like. Illustratively,acylamino includes acylmethylamino, acylethylamino, and the like.

As used herein, the term “amino and derivatives thereof” includes aminoas described herein, and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino,cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino,arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino,acylamino, and the like, each of which is optionally substituted. Theterm “amino derivative” also includes urea, carbamate, and the like.

As used herein, the term “hydroxy and derivatives thereof” includes OH,and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy,heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy,cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy,arylalkynyloxy, acyloxy, and the like, each of which is optionallysubstituted. The term “hydroxy derivative” also includes carbamate, andthe like.

As used herein, the term “thio and derivatives thereof” includes SH, andalkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio,heteroalkynylthio, cycloalkylthio, cycloalkenylthio,cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio,arylalkenylthio, arylalkynylthio, acylthio, and the like, each of whichis optionally substituted. The term “thio derivative” also includesthiocarbamate, and the like.

As used herein, the term “acyl” includes formyl, and alkylcarbonyl,alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl,heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl,cycloalkenylcarbonyl, cycloheteroalkylcarbonyl,cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl,arylalkenylcarbonyl, arylalkynylcarbonyl, acylcarbonyl, and the like,each of which is optionally substituted.

As used herein, the term “carboxylate and derivatives thereof” includesthe group CO₂H and salts thereof, and esters and amides thereof, and CN.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,nitro, sulfonic acids and derivatives thereof, carboxylic acids andderivatives thereof, and the like. Illustratively, any of amino,hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl,arylheteroalkyl, and/or sulfonic acid is optionally substituted.

As used herein, the term “optionally substituted aryl” includes thereplacement of hydrogen atoms with other functional groups on the arylthat is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,nitro, sulfonic acids and derivatives thereof, carboxylic acids andderivatives thereof, and the like. Illustratively, any of amino,hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl,arylheteroalkyl, and/or sulfonic acid is optionally substituted. Theterm “prodrug” as used herein generally refers to any compound that whenadministered to a biological system generates a biologically activecompound as a result of one or more spontaneous chemical reaction(s),enzyme-catalyzed chemical reaction(s), and/or metabolic chemicalreaction(s), or a combination thereof. In vivo, the prodrug is typicallyacted upon by an enzyme (such as esterases, amidases, phosphatases, andthe like), simple biological chemistry, or other process in vivo toliberate or regenerate the more pharmacologically active drug. Thisactivation may occur through the action of an endogenous host enzyme ora non-endogenous enzyme that is administered to the host preceding,following, or during administration of the prodrug. Additional detailsof prodrug use are described in U.S. Pat. No. 5,627,165; and Pathalk,2000. It is appreciated that the prodrug is advantageously converted tothe original drug as soon as the goal, such as targeted delivery,safety, stability, and the like is achieved, followed by the subsequentrapid elimination of the released remains of the group forming theprodrug.

Prodrugs may be prepared from the compounds described herein byattaching groups that ultimately cleave in vivo to one or morefunctional groups present on the compound, such as —OH—, —SH, —CO₂H,—NR₂. Illustrative prodrugs include but are not limited to carboxylateesters where the group is alkyl, aryl, aralkyl, acyloxyalkyl,alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and amineswhere the group attached is an acyl group, an alkoxycarbonyl,aminocarbonyl, phosphate or sulfate. Illustrative esters, also referredto as active esters, include but are not limited to 1-indanyl,N-oxysuccinimide; acyloxyalkyl groups such as acetoxymethyl,pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,β-(cyclohexylcarbonyl oxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl,and the like; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl,β-ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl groups,including di-lower alkylamino alkyl groups, such as dimethylaminomethyl,dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like;2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and lactonegroups such as phthalidyl, dimethoxyphthalidyl, and the like.

Further illustrative prodrugs contain a chemical moiety, such as anamide or phosphorus group functioning to increase solubility and/orstability of the compounds described herein. Further illustrativeprodrugs for amino groups include, but are not limited to,(C₃-C₂₀)alkanoyl; halo-(C₃-C₂₀)alkanoyl; (C₃-C₂₀)alkenoyl;(C₄-C₇)cycloalkanoyl; (C₃-C₆)-cycloalkyl(C₂-C₁₆)alkanoyl; optionallysubstituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1to 3 substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with one or more of 1 to 3halogen atoms; optionally substituted aryl(C₂-C₁₆)alkanoyl, such as thearyl radical being unsubstituted or substituted by 1 to 3 substituentsselected from the group consisting of halogen, (C₁-C₃)alkyl and(C₁-C₃)alkoxy, each of which is optionally further substituted with 1 to3 halogen atoms; and optionally substituted heteroarylalkanoyl havingone to three heteroatoms selected from O, S and N in the heteroarylmoiety and 2 to 10 carbon atoms in the alkanoyl moiety, such as theheteroaryl radical being unsubstituted or substituted by 1 to 3substituents selected from the group consisting of halogen, cyano,trifluoromethanesulphonyloxy, (C₁-C₃)alkyl, and (C₁-C₃)alkoxy, each ofwhich is optionally further substituted with 1 to 3 halogen atoms. Thegroups illustrated are exemplary, not exhaustive, and may be prepared byconventional processes.

It is understood that the prodrugs themselves may not possesssignificant biological activity, but instead undergo one or morespontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s),and/or metabolic chemical reaction(s), or a combination thereof afteradministration in vivo to produce the compound described herein that isbiologically active or is a precursor of the biologically activecompound. However, it is appreciated that in some cases, the prodrug isbiologically active. It is also appreciated that prodrugs may oftenserves to improve drug efficacy or safety through improved oralbioavailability, pharmacodynamic half-life, and the like. Prodrugs alsorefer to derivatives of the compounds described herein that includegroups that simply mask undesirable drug properties or improve drugdelivery. For example, one or more compounds described herein mayexhibit an undesirable property that is advantageously blocked orminimized may become pharmacological, pharmaceutical, or pharmacokineticbarriers in clinical drug application, such as low oral drug absorption,lack of site specificity, chemical instability, toxicity, and poorpatient acceptance (bad taste, odor, pain at injection site, and thelike), and others. It is appreciated herein that a prodrug, or otherstrategy using reversible derivatives, can be useful in the optimizationof the clinical application of a drug.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill

It is also appreciated that the therapeutically effective amount,whether referring to monotherapy or combination therapy, isadvantageously selected with reference to any toxicity, or otherundesirable side effect, that might occur during administration of oneor more of the compounds described herein. Further, it is appreciatedthat the co-therapies described herein may allow for the administrationof lower doses of compounds that show such toxicity, or otherundesirable side effect, where those lower doses are below thresholds oftoxicity or lower in the therapeutic window than would otherwise beadministered in the absence of a cotherapy.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from variousamorphous, non-amorphous, partially crystalline, crystalline, and/orother morphological forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from varioushydrates and/or solvates of the compounds described herein. Accordingly,such pharmaceutical compositions that recite compounds described hereinare to be understood to include each of, or any combination of, thevarious morphological forms and/or solvate or hydrate forms of thecompounds described herein. Illustratively, compositions may include oneor more carriers, diluents, and/or excipients. The compounds describedherein, or compositions containing them, may be formulated in atherapeutically effective amount in any conventional dosage formsappropriate for the methods described herein. The compounds describedherein, or compositions containing them, including such formulations,may be administered by a wide variety of conventional routes for themethods described herein, and in a wide variety of dosage formats,utilizing known procedures (see generally, Remington: The Science andPractice of Pharmacy, (21^(st) ed., 2005)).

The term “administering” as used herein includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxicpharmaceutically-acceptable carriers, adjuvants, and vehicles.

Illustrative routes of oral administration include tablets, capsules,elixirs, syrups, and the like.

In another embodiment, the compounds described herein include thefollowing examples. The examples further illustrate additional featuresof the various embodiments of the invention described herein. However,it is to be understood that the examples are illustrative and are not tobe construed as limiting other embodiments of the invention describedherein. In addition, it is appreciated that other variations of theexamples are included in the various embodiments of the inventiondescribed herein.

EXAMPLES

General Experimental Methods. Chemicals and reagents were purchased fromcommercial suppliers and used without further purification. Anhydroussolvents were obtained as follows: pyridine and dichloromethane weredistilled from calcium hydride; tetrahydrofuran and diethyl ether weredistilled from sodium wire with benzophenone as an indicator. All othersolvents were reagent grade. All moisture sensitive reactions werecarried out in oven dried glassware under argon. ¹H NMR and ¹³C NMRspectra were recorded on a Bruker Avance ARX-400, Bruker DRX-500, orBruker Avance-III-800 spectrometer. Chemical shifts are given in ppm andare referenced against the diluting solvent. For chloroform-d: ¹³Ctriplet=77.00 CDCl₃ and ¹H singlet=7.26 ppm. For methanol-d₄: ¹³Cseptuplet=49.05 and ¹H quintuplet=3.31 ppm. Characteristic splittingpatterns due to spin spin coupling are expressed as follows: br=broad,s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, sept=septuplet.All coupling constants are measured in hertz. FTIR spectra were recordedon a Mattson Genesis II FT-IR spectrometer or a Perkin Elmerspectrometer #L1185247 using a NaCl plate or KBr pellot. Opticalrotations were recorded on a Perkin Elmer 341 or Rudolph ResearchAutopol III polarimeter. Low resolution mass spectra were recorded on aFinniganMAT LCQ or Hewlett-Packard Engine mass spectrometer. Highresolution mass spectra were recorded on a FinniganMAT XL95 massspectrometer calibrated against PPG. Column chromatography was performedwith Whatman 240-400 mesh silica gel under low pressure of 3-5 psi. TLCwas carried out with E. Merck silica gel 60-F-254 plates. Visualizationwas carried out with short-wave UV or staining with phosphomolybdic acid(PMA), iodine, or ninhydrin. HPLC data was collected using a systemcomposed of an Agilent 1100 series degasser, quaternary pump,thermostatable column compartment, variable wavelength detector, andAgilent 1200 series autosampler and fraction collector controlled byChemstation software. All chromatographic reagents used were HPLC grade.The reported inhibitors were found to be >95% pure by reversed-phasegradient HPLC (see supporting information for specific methodconditions).

Example 1 1-(Hex-5-enyloxy)-3-methoxybenzene (5a)

To a stirred solution of 3-methoxy phenol (1.24 g, 10 mmol),5-hexen-1-ol, 4a (1.4 mL, 12 mmol) and Ph₃P (3.14 g, 12 mmol) in THF (20mL) at 0° C. was added diisopropylazodicarboxylate (2.3 mL, 12 mmol)dropwise. After stirring the solution for 30 min at 0° C. the reactionmixture was warmed to 23° C. and stirred for 3 h. The reaction mixturewas concentrated in vacuo and the residue was subjected to columnchromatography (98:2 hexanes:EtOAc) to yield 1.98 g of 5a (96% yield) asa colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 1.58-1.66 (m, 2H), 1.80-1.88(m, 2H), 2.15-2.20 (m, 2H), 3.82 (s, 3H), 3.98 (t, J=6.4 Hz, 2H),5.02-5.12 (m, 2H), 5.83-5.92 (m, 1H), 6.52-6.56 (m, 3H), 7.19-7.24 (m,1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.3, 28.7, 33.4, 55.1, 67.6, 100.9,106.0, 106.6, 114.7, 129.8, 138.5, 160.3, 160.8; FT-IR (film, NaCl)v_(max)=3075, 2939, 1599, 1493, 1287, 1200, 1152, 1046 cm⁻¹; CI LRMS m/z(ion): 207.25 (M+H)⁺.

Example 2 1-Methoxy-3-(pent-4-enyloxy)benzene (5b)

Title compound was obtained from 4-penten-1-ol 4b, as described for 5ain 95% yield after flash-chromatography (98:2 hexanes:EtOAc) as acolorless oil. ¹H NMR (400 MHz, CDCl₃): δ 1.88-1.94 (m, 2H), 2.25-2.30(n, 2H), 3.81 (s, 3H), 3.98 (t, J=6.4 Hz, 2H), 5.03-5.13 (m, 2H),5.85-5.95 (m, 1H), 6.51-6.56 (m, 3H), 7.20 (t, J=8.1 Hz, 1H); ¹³C NMR(100 MHz, CDCl₃): δ 28.3, 30.0, 55.1, 67.0, 100.9, 106.0, 106.6, 115.1,129.7, 137.7, 160.2, 160.7; FT-IR (film, NaCl) v_(max)=3076, 2940, 1599,1492, 1287, 1200, 1152, 1048 cm⁻¹; CI LRMS m/z (ion): 193.25 (M+H)⁺.

Example 3 1-(But-3-enyloxy)-3-methoxybenzene (5c)

Title compound was obtained from 3-buten-1-ol 4c, as described for 5a in96% yield after flash-chromatography (98:2 hexanes:EtOAc) as a colorlessoil. ¹H NMR (400 MHz, CDCl₃): δ 2.54-2.60 (m, 2H), 3.81 (s, 3H), 4.02(t, J=6.7 Hz, 2H), 5.13-5.23 (m, 2H), 5.89-5.97 (m, 1H), 6.51-6.56 (m,3H), 7.20 (t, J=8.1 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 33.6, 55.1,67.1, 100.9, 106.2, 106.6, 116.9, 129.8, 134.4, 160.1, 160.8; FT-IR(film, NaCl) v_(max)=3136, 2378, 1644, 1509 cm⁻¹; CI LRMS m/z (ion):179.20 (M+H)⁺.

Example 4 1-(Allyloxy)-3-methoxybenzene (5d)

Title compound was obtained from allyl alcohol 4d, as described for 5ain 96% yield after flash-chromatography (98:2 hexanes:EtOAc) as acolorless oil. ¹H NMR (400 MHz, CDCl₃): δ 3.81 (s, 3H), 4.02 (d, J=6.7Hz, 2H), 5.13-5.23 (m, 2H), 5.89-5.97 (m, 1H), 6.51-6.56 (m, 3H), 7.20(t, J=8.1 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 55.1, 67.1, 100.9, 106.2,106.6, 116.9, 129.8, 134.4, 160.1, 160.8.

Example 5 2-(Hex-5-enyloxy)-4-methoxybenzenesulfonic acid (6a)

To 6a (2 g, 9.7 mmol) was added acetic anhydride (1.4 mL, 14.5 mmol) andthe resulting mixture was stirred at 0° C. To this was then added concdH₂SO₄ (1.1 gm) followed by methanol (20 mL). The resulting solution waswarmed to 23° C. and stirred for 12 h. After this time the reactionmixture was concentrated in vacuo and the resulting red oil wassubjected to column chromatography (88:12 CH₂Cl₂:MeOH) to give 6a (1.06g, 38%) as a red waxy solid. ¹H NMR (400 MHz, D₂O): δ 1.44 (quintet,J=7.4 Hz, 2H), 1.66-1.73 (m, 2H), 1.99 (q, J=7.1 Hz, 2H), 3.70 (s, 3H),3.98 (t, J=6.5 Hz, 2H), 4.85-4.97 (m, 2H), 5.74-5.92 (m, 1H), 6.52-6.56(m, 3H), 7.19-7.24 (m, 1H); ¹³C NMR (100 MHz, D₂O): δ 25.3, 28.7, 33.4,55.1, 67.6, 100.9, 106.0, 106.6, 114.7, 129.8, 138.5, 160.3, 160.8; ESIm/z (ion): 285.09 (M−H)⁻.

Example 6 4-Methoxy-2-(pent-4-enyloxy)benzenesulfonic acid (6b)

Title compound was obtained from ether 5b as described for 6a in 36%yield after flash-chromatography (88:12 CH₂Cl₂:MeOH) as a white solid.¹H NMR (400 MHz, D₂O): δ 1.75-1.82 (m, 2H), 2.10-2.16 (m, 2H), 3.69 (s,3H), 3.98 (t, J=6.4 Hz, 2H), 4.88-5.00 (m, 2H), 5.77-5.87 (m, 1H), 6.45(dd, J=8.6, 2.2 Hz, 1H), 6.51 (d, J=2.1 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H);¹³C NMR (100 MHz, D₂O): δ 28.0, 29.9, 56.1, 68.7, 100.5, 104.9, 115.5,123.7, 130.2, 139.4, 157.8, 163.5; ESI m/z (ion): 271.07 (M−H)⁻.

Example 7 2-(But-3-enyloxy)-4-methoxybenzenesulfonic acid (6c)

Title compound was obtained from ether 5c as described for 6a in 30%yield after flash-chromatography (88:12 CH₂Cl₂:MeOH) as a white solid.¹H NMR (400 MHz, D₂O): δ 2.38-2.43 (m, 2H), 3.62 (s, 3H), 3.98 (t, J=6.8Hz, 2H), 4.93-5.05 (m, 2H), 5.77-5.87 (m, 1H), 6.37 (dd, J=8.6, 2.2 Hz,1H), 6.43 (d, J=2.4 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz,D₂O): δ 33.3, 56.1, 68.8, 100.6, 117.5, 123.7, 130.2, 135.4, 157.5,163.4; ESI m/z (ion): 257.10 (M−H)⁻.

Example 8 2-(Allyloxy)-4-methoxybenzenesulfonic acid (6d)

Title compound was obtained from ether 5d as described for 6a in 35%yield after flash-chromatography (88:12 CH₂Cl₂:MeOH) as a white solid.¹H NMR (400 MHz, D₂O): δ 2.38-2.43 (m, 2H), 3.62 (s, 3H), 3.98 (t, J=6.8Hz, 2H), 4.93-5.05 (m, 2H), 5.77-5.87 (m, 1H), 6.37 (dd, J=8.6, 2.2 Hz,1H), 6.43 (d, J=2.4 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz,D₂O): δ 33.3, 56.1, 68.8, 100.6, 117.5, 123.7, 130.2, 135.4, 157.5,163.4; ESI m/z (ion): 243.13 (M−H)⁻.

Example 9 2-(Hex-5-enyloxy)-4-methoxybenzene-1-sulfonyl chloride (7a)

To a stiffing solution of sulfonic acid 6a (266 mg, 0.9 mmol) inpyridine (2 mL) was added thionyl chloride (0.2 mL, 2.8 mmol) dropwise.The resulting solution was allowed to stir for 4 h and then the reactionmixture concentrated in vacuo. The resulting residue was purified usingcolumn chromatography (5:1 hexanes:EtOAc) to give 7a (140 mg, 50%) as acolorless oil. ¹H NMR (400 MHz, CDCl₃): δ 1.63-1.70 (m, 2H), 1.87-1.93(m, 2H), 2.10-2.16 (m, 2H), 3.88 (s, 3H), 4.14 (t, J=6.2 Hz, 2H),4.95-5.05 (m, 2H), 5.76-5.86 (m, 1H), 6.51-6.54 (m, 3H), 7.84 (d, J=9.6Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 24.9, 28.1, 33.1, 55.9, 69.2, 99.9,104.6, 114.7, 124.3, 131.7, 138.3, 158.7, 166.8.

Example 10 4-Methoxy-2-(pent-4-enyloxy)benzene-1-sulfonyl chloride (7b)

Title compound was obtained from ether 6b as described for 7a in 48%yield after flash-chromatography (6:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.96-1.2.03 (m, 2H), 2.31-2.37 (m, 2H), 3.88(s, 3H), 4.15 (t, J=6.2 Hz, 2H), 4.99-5.09 (m, 2H), 5.79-5.90 (m, 1H),6.51-6.54 (m, 3H), 7.84 (d, J=9.2 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ27.8, 29.7, 55.9, 68.4, 99.9, 104.6, 115.6, 124.3, 131.7, 137.3, 158.6,166.8.

Example 11 2-(But-3-enyloxy)-4-methoxybenzene-1-sulfonyl chloride (7c)

Title compound was obtained from ether 6c as described for 7a in 52%yield after flash-chromatography (6:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.96-1.2.03 (m, 2H), 2.31-2.37 (m, 2H), 3.88(s, 3H), 4.15 (t, J=6.2 Hz, 2H), 4.99-5.09 (m, 2H), 5.79-5.90 (m, 1H),6.51-6.54 (m, 2H), 7.84 (d, J=9.2 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ27.8, 29.7, 55.9, 68.4, 99.9, 104.6, 115.6, 124.3, 131.7, 137.3, 158.6,166.8.

Example 12 2-(Allyloxy)-4-methoxybenzene-1-sulfonyl chloride (7d)

Title compound was obtained from ether 6d as described for 7a in 58%yield after flash-chromatography (6:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 3.88 (s, 3H), 4.72 (d, J=4.4 Hz, 2H), 5.33(d, J=10.6 Hz, 1H), 5.57 (d, J=17.3 Hz, 1H), 5.99-6.07 (m, 1H),6.52-6.55 (m, 2H), 7.83 (d, J=8.7 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ55.9, 69.7, 100.5, 105.0, 118.1, 124.4, 131.0, 131.7, 158.0, 166.7.

Example 13 tert-Butyl(2S,3R)-4-(hex-5-enylamino)-3-hydroxy-1-phenylbutan-2-ylcarbamate (10a)

A solution of hex-5-en-1-amine 9a (297 mg, 3 mmol) and epoxide 8 (263mg, 1 mmol) was heated to 60° C. in isopropanol (4 mL) for 4 h. Thesolvent was then evaporated under reduced pressure and the resultingresidue was purified by silica chromatography (5:95 MeOH:CHCl₃) to give10a (350 mg, 97%) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 1.34 (s,9H), 1.39-1.50 (m, 4H), 2.05 (q, J=7 Hz, 2H), 2.55-2.68 (m, 6H),2.81-2.86 (m, 1H), 2.96 (dd, J=4.5, 14 Hz, 1H), 3.44-3.49 (m, 1H), 3.79(br s, 1H), 4.72 (d, J=8.7 Hz, 1H), 4.92-5.02 (m, 2H), 5.74-5.84 (m,1H), 7.17-7.28 (m, 5H); ¹³C NMR (100 MHz, CDCl₃): δ 26.4, 28.2, 29.4,33.4, 36.5, 49.6, 51.3, 54.1, 70.7, 114.5, 126.2, 128.3, 129.4, 137.8,138.6, 155.9; CI LRMS m/z (ion): 363.55 (M+

Example 14 tert-Butyl(2S,3R)-4-(allylamino)-3-hydroxy-1-phenylbutan-2-ylcarbamate (10b)

Title compound was obtained from allylamine 9b and epoxide 8 asdescribed for 10a in 99% yield after flash-chromatography (5:95MeOH:CHCl₃) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 1.35 (s, 9H),2.60-2.86 (m, 5H), 2.96 (dd, J=4.5, 14.1 Hz, 1H), 3.16-3.29 (m, 2H),3.50-3.53 (m, 1H), 3.81 (br s, 1H), 4.73 (d, J=9.1 Hz, 1H), 5.10 (d,J=10.3 Hz, 1H), 5.17 (d, J=17 Hz, 1H), 5.81-5.91 (m, 1H), 7.13-7.32 (m,5H); ¹³C NMR (100 MHz, CDCl₃): δ 28.2, 36.5, 50.7, 52.1, 54.1, 70.9,79.3, 116.2, 126.2, 128.3, 129.4, 137.8, 155.9; CI LRMS m/z (ion) 321.50(M+H)⁺.

Example 15tert-Butyl-(2S,3R)-4-(N-(hex-5-enyl)-2-(hex-5-enyloxy)-4-ethoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11a)

To a stirring solution of 10a (50 mg, 0.14 mmol) in pyridine (2 mL) wasadded 7a (64 mg, 0.20 mmol) and the resulting solution was allowed tostir for 2 h at 23° C. The reaction mixture was concentrated underreduced pressure and the resulting residue was purified by flash columnchromatography (3:1 hexanes:EtOAc) to yield 74 mg (84% yield) of 11a asa colorless oil. [α]_(D) ²⁰ −1.4 (c 1.00, CHCl₃); ¹H NMR (400 MHz,CDCl₃): δ 1.25-1.34 (m, 12H), 1.47-1.50 (m, 2H), 1.57-1.60 (m, 2H),1.82-1.90 (m, 2H), 1.97 (q, J=7.1 Hz, 2H), 2.11 (q, J=7.0 Hz, 2H),2.92-2.95 (m, 2H), 3.10-3.17 (m, 1H), 3.30 (br s, 3H), 3.76 (br s, 2H),3.84 (s, 3H), 3.90-3.92 (m, 1H) 4.03 (t, J=6.7 Hz, 2H), 4.65 (d, J=7.1Hz, 1H), 4.89-5.04 (m, 4H), 5.66-5.82 (m, 2H), 6.46 (d, J=2 Hz), 6.49(dd, J=8.8, 2.3 Hz, 1H), 7.17-7.29 (m, 5H), 7.83 (d, J=7.8 Hz, 1H); ¹³CNMR (100 MHz, CDCl₃): δ 24.9, 25.7, 27.9, 28.2, 28.3, 33.1, 33.2, 35.1,49.9, 52.3, 54.5, 55.6, 69.1, 72.2, 79.4, 100.2, 104.1, 114.7, 115.0,119.4, 126.2, 128.3, 129.5, 133.5, 137.8, 138.1, 138.2, 156.0, 157.6,164.7; FT-IR (film, NaCl) v_(max)=3395, 2935, 1705, 1597, 1325 cm⁻¹; ESI(+) LRMS m/z (ion): 653.13 (M+Na)⁺.

Example 16tert-Butyl-(2S,3R)-4-(N-(hex-5-enyl)-4-methoxy-2-(pent-4enyloxy)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11b)

Title compound was obtained from 10a and 7b, as described for 11a in 79%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.[α]_(D) ²⁰ −1.6 (c 1.20, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 1.25-1.37(m, 12H), 1.43-1.51 (m, 2H), 1.88-1.98 (m, 4H), 2.27 (q, J=7.1 Hz, 2H),2.86-2.97 (m, 2H), 3.10-3.17 (m, 1H), 3.25-3.31 (m, 3H), 3.76 (br s,2H), 3.84 (s, 3H), 4.04 (t, J=6.6 Hz, 2H), 4.66 (d, J=7.1 Hz, 1H),4.88-5.10 (m, 4H), 5.65-5.72 (m, 1H), 5.78-5.85 (m, 1H), 6.46 (d, J=2Hz), 6.49 (dd, J=8.8, 2.0 Hz, 1H), 7.17-7.29 (m, 5H), 7.83 (d, J=8.7 Hz,1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.7, 27.9, 28.2, 33.1, 35.2, 49.8,52.2, 54.6, 55.6, 68.4, 72.2, 79.4, 100.2, 104.1, 114.7, 115.7, 119.4,126.2, 128.3, 129.5, 133.5, 137.0, 137.8, 138.2, 156.0, 157.6, 164.7;FT-IR (film, NaCl) v_(max)=3398, 2931, 1706, 1596, 1325 cm⁻¹; ESI (+)LRMS m/z (ion): 639.06 (M+Na)⁺.

Example 17tert-Butyl-(2S,3R)-4-(2-(but-3-enyloxy)-N-(hex-5-enyl)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11c)

Title compound was obtained from 10a and 7c, as described for 11a in 50%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.[α]_(D) ²⁰+0.6 (c 2.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.28-1.38 (m,12H), 1.48-1.52 (m, 2H), 2.00 (q, J=6.7 Hz, 2H), 2.65 (q, J=6.7, 2H),2.96 (br s, 2H), 3.14-3.18 (m, 1H), 3.30-3.39 (m, 3H), 3.79 (br s, 2H),3.88 (s, 3H), 4.13 (t, J=7.1 Hz, 2H), 4.68 (d, J=5.1 Hz, 1H), 4.92-4.98(m, 2H), 5.17-5.24 (m, 2H), 5.68-5.76 (m, 1H), 5.91-5.99 (m, 1H), 6.51(d, J=2 Hz, 1H), 6.54 (dd, J=8.8, 2.0 Hz, 1H), 7.21-7.32 (m, 5H), 7.88(d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 25.9, 28.4, 33.4, 35.4,50.1, 52.5, 54.8, 55.9, 68.7, 72.5, 79.7, 100.6, 104.5, 114.9, 118.0,119.8, 126.5, 128.6, 129.8, 133.7, 133.9 138.1, 138.5, 156.3, 157.7,165.0; FT-IR (film, NaCl) v_(max)=3394, 2931, 1704, 1596, 1325 cm⁻¹; ESI(+) m/z (ion): 625.05 (M+Na)⁺.

Example 18tert-Butyl(2S,3R)-4-(2-(allyloxy)-N-(hex-5-enyl)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11d)

Title compound was obtained from 10a and 7d, as described for 11a in 64%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.[α]_(D) ²⁰+1.1 (c 2.80, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 1.25-1.34 (m,12H), 1.39-1.46 (m, 2H), 1.96 (q, J=7.0 Hz, 2H), 2.89-2.96 (m, 2H),3.08-3.15 (m, 1H), 3.24-3.30 (m, 3H), 3.77 (br s, 2H), 3.84 (s, 3H),4.60 (d, J=5.4 Hz, 2H), 4.66 (d, J=7.2 Hz, 1H), 4.88-4.94 (m, 2H), 5.33(d, J=10.4, 2H), 5.43 (d, J=17.4 Hz, 1H), 5.63-5.73 (m, 1H), 6.00-6.10(m, 1H), 6.47 (d, J=1.8 Hz, 1H), 6.54 (dd, J=8.9, 1.9 Hz, 1H), 7.18-7.29(m, 5H), 7.85 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.7, 28.2,33.1, 35.2, 49.9, 52.5, 54.5, 55.6, 69.9, 72.2, 79.4, 100.6, 104.4,114.7, 119.4, 119.7 126.2, 128.3, 129.5, 131.9, 133.5, 137.9, 138.2,155.9, 157.0, 164.6; FT-IR (film, NaCl) v_(max)=3400, 2929, 1704, 1596,1324 cm⁻¹; ESI (+) m/z (ion): 611.03 (M+Na)⁺.

Example 19tert-Butyl-(2S,3R)-4-(N-allyl-2-(hex-5-enyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11e)

Title compound was obtained from 10b and 7a, as described for 11a in 77%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.[α]_(D) ²⁰ −6.6 (c 1.96, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.36 (s,9H), 1.60-1.66 (m, 2H), 1.87-1.97 (m, 2H), 2.13-2.17 (m, 2H), 2.91-2.98(m, 2H), 3.27-3.39 (m, 2H), 3.79 (br s, 2H), 3.87 (s, 3H), 3.91-3.99 (m,2H), 4.08 (t, J=6.7 Hz, 2H), 4.65 (d, J=7.1 Hz, 1H), 4.98 (d, J=10.1,1H), 5.04 (d, J=17.1, 1H), 5.13-5.19 (m, 2H), 5.63-5.73 (m, 1H),5.78-5.86 (m, 1H), 6.50-6.53 (m, 2H), 7.21-7.31 (m, 5H), 7.88 (d, J=8.7Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.0, 28.3, 33.3, 35.3, 51.2, 52.2,54.6, 55.7, 69.2, 71.8, 79.5, 100.3, 104.2, 115.1, 119.0, 119.6, 126.3,128.4, 129.6, 133.6, 137.9, 138.2, 156.1, 157.7, 164.8; FT-IR (film,NaCl) v_(max)=3392, 2932, 1702, 1595, 1324 cm⁻¹; ESI (+) m/z (ion):611.04 (M+Na)⁺.

Example 20tert-Butyl-(2S,3R)-4-(N-allyl-4-methoxy-2-(pent-4-enyloxy)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11f)

Title compound was obtained from 10b and 7b, as described for 11a in 86%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.[α]_(D) ²⁰ −5.6 (c 1.10, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 1.32 (s,9H), 1.94 (quintet, J=7 Hz, 2H), 2.65 (q, J=7 Hz, 2H), 2.86-2.96 (m,2H), 3.27 (dd, J=7.4, 14.8 Hz, 1H), 3.34-3.38 (m, 1H), 3.76 (br s, 2H),3.82 (s, 3H), 3.87-3.92 (m, 2H), 4.04 (t, J=6.5 Hz, 2H), 4.65 (d, J=8.2Hz, 1H), 4.98-5.15 (m, 4H), 5.57-5.63 (m, 1H), 5.75-5.86 (m, 1H),6.46-6.49 (m, 2H), 7.12-7.27 (m, 5H), 7.83 (d, J=8.5 Hz, 1H); ¹³C NMR(100 MHz, CDCl₃): δ 27.8, 28.1, 35.3, 51.0, 51.9, 54.5, 55.6, 68.4,71.8, 79.3, 100.2, 104.2, 115.7, 119.0, 119.5, 126.2, 128.2, 129.5,133.4, 137.2, 137.9, 155.9, 157.6, 164.7; FT-IR (film, NaCl)v_(max)=3390, 2976, 1710, 1597, 1325 cm⁻¹; ESI (+) m/z (ion): 597.13(M+Na)⁺.

Example 21tert-Butyl-(2S,3R)-4-(N-allyl-2-(but-3-enyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11g)

Title compound was obtained from 10b and 7c, as described for 11a in 78%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.¹H NMR (500 MHz, CDCl₃): δ 1.34 (s, 9H), 2.62 (q, J=7 Hz, 2H), 2.88-2.96(m, 2H), 3.27 (dd, J=7.8, 15.1 Hz, 1H), 3.34-3.38 (m, 1H), 3.76 (br s,2H), 3.85 (s, 3H), 3.87-3.99 (m, 2H), 4.10 (t, J=6.5 Hz, 2H), 4.65 (brs, 1H), 5.10-5.22 (m, 4H), 5.57-5.63 (m, 1H), 5.87-5.95 (m, 1H), 6.49(d, J=2.2 Hz, 1H), 6.51 (dd, J=2.3, 8.7 Hz 1H) 7.18-7.37 (m, 5H), 7.85(d, J=8.7 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): 27.8, 28.1, 35.3, 51.0,51.9, 54.5, 55.6, 68.4, 71.8, 79.3, 100.2, 104.2, 115.7, 119.0, 119.5,126.2, 128.2, 129.5, 133.4, 137.2, 137.9, 155.9, 157.6, 164.7; FT-IR(film, NaCl) v_(max)=3390, 2976, 1710, 1597, 1325 cm⁻¹; ESI (+) m/z(ion): 582.95 (M+Na)⁺.

Example 22tert-Butyl-(2S,3R)-4-(N-allyl-2-(allyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(11h)

Title compound was obtained from 10b and 7d, as described for 11a in 80%yield after flash-chromatography (3:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.33 (s, 9H), 2.83-2.96 (m, 2H), 3.25-3.36(m, 2H), 3.77 (br s, 2H), 3.83 (s, 3H), 3.87-3.94 (m, 2H), 4.56-4.67 (m,3H), 5.07-5.14 (m, 2H), 5.33 (d, J=10.5 Hz, 1H), 5.44 (d, J=17.2 Hz,1H), 5.57-5.64 (m, 1H), 6.00-6.10 (m, 1H), 6.47 (d, J=1.9 Hz, 1H), 6.51(dd, J=1.9, 8.9 Hz, 1H) 7.16-7.28 (m, 5H), 7.85 (d, J=8.7 Hz, 1H); ¹³CNMR (100 MHz, CDCl₃): δ 28.2, 35.3, 51.3, 52.1, 54.5, 55.6, 69.9, 71.8,79.3, 100.6, 104.5, 119.0, 119.4, 119.8, 126.2, 128.2, 129.5, 131.8,133.4, 137.9, 155.9, 157.0, 164.7; FT-IR (film, NaCl) v_(max)=3390,2976, 1710, 1597, 1325 cm⁻¹; ESI (+) m/z (ion): 569.06 (M+Na)⁺.

Example 23 Compound 13a

To a stirring solution of 11a (63 mg, 0.1 mmol) in CH₂Cl₂ (2 mL) wasadded a solution of 30% trifluoroacetic acid in CH₂Cl₂ and the resultingmixture was stirred for 30 min. The solvent was evaporated under reducedpressure and the residue was dissolved in CH₃CN (2 mL). The solution wasadded to 12 (31 mg, 0.11 mmol), followed by i-Pr₂NEt. After stirring for24 h the reaction mixture was concentrated in vacuo and the resultingresidue was subjected to flash-chromatography (1:1 hexanes:EtOAc) togive 36 mg (53% yield) of 13a as a colorless oil. [α]_(D) ²⁰ −13.1 (c1.50, CHCl₃); ¹H NMR (500 MHz, CDCl₃): δ 1.28-1.31 (m, 2H), 1.47-1.51(m, 2H), 1.58-1.61 (m, 3H), 1.84-1.88 (m, 2H), 1.96-1.99 (m, 2H),2.01-2.13 (m, 2H), 2.79-2.84 (m, 1H), 2.98-3.02 (m, 1H), 3.09-3.15 (m,1H), 3.24-3.40 (m, 3H), 3.61 (br s, 1H), 3.64-3.72 (m, 2H), 3.83-3.92(m, 4H), 3.93-3.96 (m, 1H), 4.05 (t, J=6.7 Hz, 2H), 4.90-5.03 (m, 4H),5.64 (d, J=5 Hz, 1H), 5.66-5.82 (m, 2H), 6.46 (s, 1H), 6.51 (d, J=8.8Hz, 1H), 7.17-7.26 (m, 5H), 7.83 (d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 27.9, 28.2, 28.1, 29.4, 31.7, 32.9, 33.1, 35.1, 45.1, 49.8,52.1, 54.8, 55.5, 68.9, 69.3, 70.5, 72.0 73.1, 100.1, 103.9, 109.0,114.6, 114.9, 118.9, 126.3, 128.2, 128.7, 129.1, 133.4, 137.4, 137.8,138.0, 155.2, 157.4, 164.6; FT-IR (film, NaCl) v_(max)=3343, 2928, 1721,1595, 1325 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₆H₅₀N₂O₉S,709.3135. found, 709.3136.

Example 24 Compound 13b

Title compound was obtained from 11b and 12 as described for 13a in 55%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (500 MHz, CDCl₃): δ 1.27-1.32 (m, 2H), 1.41-1.53 (m, 3H),1.57-1.62 (m, 1H), 1.92-1.99 (m, 4H), 2.80 (dd, J=10, 14 Hz, 1H),2.86-2.91 (m, 1H), 3.01 (dd, J=4, 14 Hz, 1H), 3.10-3.15 (m, 1H),3.27-3.33 (m, 2H), 3.38 (dd, J=8.6, 15.2 Hz, 1H), 3.61 (br s, 1H),3.64-3.70 (m, 2H), 3.81-3.84 (m, 5H), 3.88-3.95 (m, 2H), 4.05 (t, J=6.6Hz, 2H), 4.89-5.09 (m, 4H), 5.63 (d, J=5.2 Hz, 1H), 5.65-5.73 (m, 1H),5.77-5.85 (m, 1H), 6.46 (d, J=2 Hz, 1H), 6.51 (dd, J=2.1, 8.8 Hz, 1H),7.17-7.26 (m, 5H), 7.83 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ25.7, 27.9, 29.7, 33.1, 35.4, 45.1, 49.8, 52.1, 55.1, 55.7, 68.5, 69.5,70.7, 72.2 73.2, 100.3, 104.2, 109.2, 114.8, 115.7, 119.2, 126.4, 128.4,129.3, 133.6, 137.1, 137.6, 138.2, 155.4, 157.5, 164.8; ESI (+) HRMS(m/z): [M+H]⁺ calcd for C₃₅H₄₈N₂O₉S, 673.3159. found, 673.3153.

Example 25 Compound 13c

Title compound was obtained from 11c and 12 as described for 13a in 81%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (500 MHz, CDCl₃): δ 1.31-1.35 (m, 2H), 1.50-1.54 (m, 3H),1.62-1.67 (m, 1H), 2.00 (q, J=7 Hz, 2H), 2.65 (q, J=6.7 Hz, 2H),2.82-2.87 (m, 1H), 2.91-2.94 (m, 1H), 3.06 (dd, J=4.1, 14.2 Hz, 1H),3.13-3.19 (m, 1H), 3.30-3.43 (m, 3H), 3.62 (br s, 1H), 3.68-3.74 (m,2H), 3.85-3.88 (m, 4H), 3.92-3.99 (m, 2H), 4.13 (t, J=6.9 Hz, 2H),4.93-5.05 (m, 2H), 5.17-5.24 (m, 2H), 5.66 (d, J=5.1 Hz, 1H), 5.69-5.77(m, 1H), 5.90-5.99 (m, 1H), 6.52 (s, 1H), 6.55 (dd, J=2.05, 8.8 Hz, 1H),7.22-7.30 (m, 5H), 7.87 (d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ25.8, 28.1, 29.8, 33.3, 35.6, 45.4, 50.0, 52.4, 55.2, 55.8, 68.7, 69.7,70.8, 72.4, 73.4, 100.6, 104.5, 109.4, 114.9, 119.5, 126.6, 128.6,129.5, 133.5, 137.8, 138.3, 155.9, 157.6, 164.9; FT-IR (film, NaCl)v_(max)=3339, 2929, 1719, 1596, 1324 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺calcd for C₃₄H₄₆N₂O₉S, 681.2822. found, 681.2812.

Example 26 Compound 13d

Title compound was obtained from 11d and 12 as described for 13a in 55%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (500 MHz, CDCl₃): δ 1.24-1.30 (m, 2H), 1.43-1.51 (m, 3H),1.57-1.63 (m, 1H), 1.96 (q, J=7 Hz, 2H), 2.80 (dd, J=10, 14 Hz, 1H),2.86-2.91 (m, 1H), 3.01 (dd, J=4.5, 14.5 Hz, 1H), 3.08-3.14 (m, 1H),3.25-3.30 (m, 2H), 3.40 (dd, J=8.5, 15 Hz, 1H) 3.58 (br s, 1H),3.65-3.72 (m, 2H), 3.80-3.82 (m, 2H), 3.87 (s, 3H), 3.88-3.96 (m, 2H),4.60 (d, J=5.5 Hz, 2H), 4.90-4.95 (m, 2H), 4.98-5.06 (m, 2H), 5.33 (d,J=10 Hz, 1H), 5.45 (d, J=18 Hz, 1H), 5.64 (d, J=5.5 Hz, 1H), 5.65-5.72(m, 1H), 6.02-6.10 (m, 1H), 6.47 (d, J=2 Hz, 1H), 6.53 (dd, J=2.5, 9 Hz,H), 7.17-7.27 (m, 5H), 7.85 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ25.7, 27.8, 33.0, 35.3, 45.2, 49.9, 52.4, 55.6, 69.5, 69.9, 70.6, 72.2,73.3, 100.6, 104.5, 109.2, 114.7, 119.4, 126.4, 128.4, 129.3, 131.9,133.5, 137.6, 138.1, 155.4, 157.0, 164.7; FT-IR (film, NaCl)v_(max)=3339, 1718, 1594, 1324 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺ calcdfor C₃₃H₄₄N₂O₉S, 667.2665. found, 667.2661.

Example 27 Compound 13e

Title compound was obtained from 11e and 12 as described for 13a in 68%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.52-1.61 (m, 3H), 1.81-1.93 (m, 2H), 2.09(q, J=7 Hz, 2H), 2.76 (dd, J=10, 13.7 Hz, 1H), 2.83-2.88 (m, 1H), 3.01(dd, J=3.6, 14.2 Hz, 1H), 3.28-3.32 (m, 2H), 3.61-3.69 (m, 2H),3.78-3.85 (m, 5H), 3.87-3.93 (m, 3H), 4.04 (t, J=6.5 Hz, 2H), 4.92-5.01(m, 2H), 5.09-5.16 (m, 2H), 5.60-5.71 (m, 2H), 5.72-5.81 (m, 1H),6.47-6.51 (m, 2H), 7.14-7.26 (m, 5H), 7.82 (d, J=8.6 Hz, 1H); ¹³C NMR(100 MHz, CDCl₃): δ 24.9, 25.7, 33.2, 35.4, 45.3, 51.0, 52.1, 54.9,55.7, 69.1, 69.5, 70.7, 71.8, 73.1, 100.3, 104.2, 109.2, 115.0, 119.0,119.2, 126.3, 128.3, 129.3, 133.4, 133.6, 137.5, 138.0, 155.4, 157.6,164.8; FT-IR (film, NaCl) v_(max)=3368, 1720, 1596, 1325 cm⁻¹; ESI (+)HRMS (m/z): [M+Na]⁺ calcd for C₃₃H₄₄N₂O₉S, 667.2665. found, 667.2668.

Example 28 Compound 13f

Title compound was obtained from 11f and 12 as described for 13a in 64%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.37-1.43 (m, 1H), 1.54-1.62 (m, 2H),1.91-1.97 (m, 2H), 2.27 (q, J=7 Hz, 2H), 2.76 (dd, J=10.1, 13.9 Hz, 1H),2.84-2.89 (m, 1H), 3.02 (dd, J=4, 14.1 Hz, 1H), 3.30-3.37 (m, 2H), 3.54(br s, 1H), 3.62-3.69 (m, 2H), 3.79-3.87 (m, 5H), 3.88-3.95 (m, 4H),4.06 (t, J=6.7 Hz, 2H), 4.96-5.00 (m, 2H), 5.04-5.16 (m, 4H), 5.59-5.67(m, 2H), 5.76-5.85 (m, 1H), 6.47 (d, J=1.9 Hz, 1H), 6.50 (dd, J=2.1, 8.9Hz, 1H), 7.16-7.26 (m, 5H), 7.82 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz,CDCl₃): δ 25.7, 27.9, 29.7, 35.5, 45.3, 51.0, 52.1, 54.9, 55.7, 68.5,69.5, 70.7, 71.8, 73.2, 100.3, 104.2, 109.2, 115.7, 119.1, 119.3, 126.4,128.3, 129.3, 133.5, 137.1, 137.7, 155.4, 157.6, 164.8; FT-IR (film,NaCl) v_(max)=3350, 1720, 1596, 1325 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺calcd for C₃₂H₄₂N₂O₉S, 653.2509. found, 653.2509.

Example 29 Compound 13g

Title compound was obtained from 11g and 12 as described for 13a in 68%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.39-1.45 (m, 1H), 1.56-1.63 (m, 1H), 2.61(q, J=6.6 Hz, 2H), 2.75-2.80 (m, 1H), 2.86-2.91 (m, 1H), 3.02 (dd,J=4.1, 14.1 Hz, 1H), 3.32 (d, J=5.8 Hz, 2H), 3.53 (br s, 1H), 3.64-3.70(m, 2H), 3.81-3.95 (m, 8H), 4.10 (t, J=6.7 Hz, 2H), 4.97-5.01 (m, 2H),5.12-5.20 (m, 4H), 5.62-5.66 (m, 2H), 5.86-5.94 (m, 1H), 6.49 (s, 1H),6.51 (d, J=9.1 Hz, 1H), 7.18-7.26 (m, 5H), 7.85 (d, J=8.8 Hz, 1H); ¹³CNMR (100 MHz, CDCl₃): 25.8, 33.2, 35.6, 45.4, 51.2, 52.3, 55.0, 55.8,68.7, 69.6, 70.8, 71.9, 73.3, 100.5, 104.5, 109.3, 117.9, 119.2, 119.4,126.5, 128.5, 129.4, 133.5, 133.7, 137.8, 155.4, 157.5, 164.9; FT-IR(film, NaCl) v_(max)=3350, 1722, 1596, 1325 cm⁻¹; ESI (+) HRMS (m/z):[M+H]⁺ calcd for C₃₁H₄₀N₂O₉S, 617.2533. found, 617.2540.

Example 30 Compound 13h

Title compound was obtained from 11h and 12 as described for 13a in 52%yield after flash-chromatography (1:1 hexanes:EtOAc) as a colorless oil.¹H NMR (400 MHz, CDCl₃): δ 1.37-1.43 (m, 1H), 1.54-1.65 (m, 1H),2.73-2.80 (m, 1H), 2.85-2.90 (m, 1H), 3.02 (dd, J=2.9, 13.7 Hz, 1H),3.28-3.39 (m, 2H), 3.51 (br s, 1H), 3.63-3.70 (m, 2H), 3.77-3.95 (m,10H), 4.62 (d, J=5.2 Hz, 2H), 4.96-5.06 (m, 2H), 5.10-5.16 (m, 2H), 5.33(d, J=10.4 Hz, 1H), 5.45 (d, J=17.2 Hz, 1H), 5.62-5.66 (m, 2H),6.01-6.10 (m, 1H), 6.49 (s, 1H), 6.52 (d, J=8.9 Hz, 1H), 7.18-7.33 (m,5H), 7.85 (d, J=8.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 25.7, 33.4,45.3, 51.4, 52.2, 54.9, 55.7, 69.6, 70.0, 70.7, 71.8, 73.1, 100.7,104.5, 109.2, 119.1, 119.4, 119.5, 126.4, 128.4, 129.3, 133.5, 137.7,155.3, 157.0, 164.8; FT-IR (film, NaCl) v_(max)=3351, 1717, 1596, 1324cm⁻¹; ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₀H₃₈N₂O₉S, 603.2376. found,603.2375.

Example 31 Inhibitor 14a

To stirring solution of 13a (32 mg, 0.047 mmol) in CH₂Cl₂ (15 mL) wasadded Grubbs 1^(st) gen. cat. (4 mg, 0.0046 mmol). After stirring at 23°C. for 16 h, the solvent was evaporated under reduced pressure and theresidue was subjected to flash column chromatography to yield 14a (27mg, 88% yield) as a white solid and E/Z mixture (3:1, determined byHPLC). The isomers were isolated by reversed-phase HPLC using thefollowing conditions: YMC Pack ODS-A column (250×100 mm, 5 micron); Flowrate=2.75 mL/min; Isocratic 60:40 CH₃CN:H₂O; T=35° C.; X=215 nm; Eisomer R₁=16.47 min; Z isomer R₁=14.45 min.

Example 32

14aE: ¹H NMR (800 MHz, CDCl₃): δ 1.39-1.44 (m, 2H), 1.48-1.53 (m, 1H),1.57-1.64 (m, 4H), 1.68-1.74 (m, 3H), 1.77-1.82 (m, 1H), 1.83-188 (m,1H), 1.94-1.98 (m, 1H), 2.10-2.14 (m, 3H), 2.71 (dd, J=9.6, 14.1 Hz,1H), 2.86-2.89 (m, 1H), 2.90 (dd, J=4.3, 14.1 Hz, 1H), 2.91-2.99 (m,2H), 3.31-3.33 (m, 1H), 3.61-3.64 (m, 1H), 3.65-3.70 (m, 2H), 3.73 (brs, 1H), 3.76-3.78 (m, 1H), 3.79-3.85 (m, 5H), 3.93 (dd, J=6.6, 9.4 Hz,1H), 3.96-3.98 (m, 1H), 4.02-4.05 (m, 1H), 4.87 (d, J=9.1 Hz, 1H),4.97-5.00 (m, 1H), 5.44-5.48 (m, 1H), 5.54-5.56 (m, 1H), 5.63 (d, J=5.1Hz, 1H), 6.44 (d, J=2 Hz, 1H), 6.49 (dd, J=2.2, 8.8 Hz, 1H), 7.13-7.24(m, 5H), 7.84 (d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 25.1, 25.3,25.7, 26.8, 29.6, 29.8, 30.4, 32.5, 35.5, 45.2, 50.6, 51.6, 54.7, 55.6,68.8, 69.5, 70.7, 71.6, 73.3, 99.9, 103.9, 109.2, 118.1, 126.4, 128.4,129.3, 130.5, 132.1, 134.0, 137.4, 155.2, 157.9, 164.9; ESI (+) HRMS(m/z): [M+Na]⁺ calcd for C₃₄H₄₆N₂O₉S, 681.2822. found, 681.2815.

Example 33

14aZ: ¹H NMR (800 MHz, CDCl₃): δ 1.28-1.33 (m, 2H), 1.38-1.47 (m, 2H),1.52-1.64 (m, 6H), 1.84-1.90 (m, 2H), 2.04-2.08 (m, 2H), 2.16-2.22 (m,2H), 2.74 (dd, J=9.5, 14.1 Hz, 1H), 2.88-2.90 (m, 1H), 3.00 (dd, J=4.5,14.1 Hz, 1H), 3.05-3.14 (m, 2H), 3.15 (dd, J=9.4, 15.1 Hz, 1H),3.44-3.48 (m, 1H), 3.61 (br s, 1H), 3.66-3.71 (m, 2H), 3.76-3.89 (m,6H), 3.95 (dd, J=6.2, 9.6 Hz, 1H), 4.10-4.15 (m, 1H), 4.87 (d, J=9.1 Hz,1H), 5.00-5.03 (m, 1H), 5.35-5.39 (m, 1H), 5.49-5.52 (m, 1H), 5.63 (d,J=5.2 Hz, 1H), 6.50 (s, 1H), 6.49 (dd, J=2.3, 8.8 Hz, 1H), 7.17-7.26 (m,5H), 7.84 (d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 25.0, 25.7,26.0, 26.6, 27.8, 29.6, 35.5, 45.2, 48.5, 52.0, 54.7, 55.6, 68.8, 69.5,70.6, 71.9, 73.3, 100.7, 104.2, 109.2, 117.8, 126.4, 128.4, 129.8,130.0, 134.1, 137.5, 155.2, 158.0, 165.0; ESI (+) HRMS (m/z): [M+Na]⁺calcd for C₃₄H₄₆N₂O₉S, 681.2822. found, 681.2819.

Example 34 Inhibitor 14b

The title compound was obtained from a ring closing metathesis reactionof 13b using Grubbs 1^(st) gen. cat. as described for 14a. The crudematerial was purified by silica chromatography (60:40 EtOAc:Hexane) togive the desired product (50% yield) as a mix of E/Z isomers (27:73 byHPLC). The isomers were isolated by chiral HPLC using the followingconditions: Chiralpak IA column (250×4.6 mm, 5 micron); Flow rate=0.75mL/min; Isocratic 60:40 IPA:Hexane; T=25° C.; X=215 nm; E isomerR_(t)=7.56 min; Z isomer R₁=8.89 min.

Example 35

14b-E: ¹H NMR (800 MHz, CDCl₃): δ 1.60-1.20 (m, 4H), 2.30-1.90 (m, 7H),3.10-2.80 (m, 5H), 3.32 (m, 1H), 4.00-3.50 (m, 12H), 4.15 (m, 2H), 4.98(m, 2H), 5.45 (m, 1H), 5.63 (m, 2H), 6.51 (m, 2H), 7.25-7.15 (m, 5H),7.76 (d, J=19.2 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 23.1, 24.8, 25.0,25.7, 26.2, 26.6, 29.0, 35.5, 45.2, 48.2, 52.0, 54.8, 55.6, 67.1, 69.5,70.7, 71.6, 73.3, 100.1, 104.0, 109.2, 114.6, 118.4, 126.4, 128.4,128.9, 129.3, 133.6, 133.8, 137.4, 155.3, 157.7, 164.9; ESI (+) HRMS(m/z): [M+Na]⁺ calcd for C₃₃H₄₄N₂O₉S, 667.2665. found, 667.2660.

Example 36

14b-Z: ¹H NMR (800 MHz, CDCl₃): δ 1.60-1.0 (m, 7H); 1.72 (m, 2H), 1.90(m, 3H), 2.21 (m, 2H), 2.43 (m, 1H), 2.55 (m, 1H), 2.67 (m, 1H), 2.76(m, 1H), 2.84 (m, 1H), 3.01 (m, 1H), 3.26 (m, 1H), 3.41 (m, 3H), 3.60(m, 4H), 3.69 (m, 1H), 3.86 (m, 2H), 4.43 (d, J=15.2 Hz, 1H), 4.83 (m,1H), 5.24 (m, 1H), 5.33 (m, 1H), 5.51 (d, J=8.8 Hz, 1H), 6.45 (m, 2H),7.17 (m, 3H), 7.24 (m, 2H), 7.87 (d, J=13.6 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 24.7, 25.7, 27.3, 28.6, 30.9, 32.1, 35.5, 45.2, 49.3, 51.8,54.8, 55.6, 69.5, 70.7, 71.0, 71.9, 73.3, 101.2, 104.3, 109.2, 119.3,126.4, 128.4, 128.9, 129.3, 130.8, 130.9, 133.4, 137.5, 155.3, 158.2,164.5; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₃H₄₄N₂O₉S, 667.2665.found, 667.2667.

Example 37 Inhibitor 14c

Title compound was obtained from 13c and Grubbs 1^(st) gen. cat. asdescribed for 14a in 89% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid and E/Z mixture (3:1, determined byHPLC). The isomers were isolated using reversed-phase HPLC under thefollowing conditions: YMC Pack ODS-A column (250×100 mm, 5 micron); Flowrate=2.75 mL/min; Isocratic 60:40 CH₃CN:H₂O; T=35° C.; X=215 nm; Eisomer R_(t)=13.43 min; Z isomer R_(t)=11.76 min.

Example 38

14cE: ¹H NMR (800 MHz, CDCl₃): δ 1.45-1.54 (m, 3H), 1.56-1.64 (m, 2H),1.68-1.72 (m, 1H), 2.11-2.20 (m, 2H), 2.52-2.62 (m, 2H), 2.79 (dd,J=9.6, 14 Hz, 1H), 2.87-2.90 (m, 1H), 2.96-3.00 (m, 2H), 3.04 (dd,J=4.2, 14.2 Hz, 1H), 3.40-3.44 (m, 1H), 3.53-3.56 (m, 1H), 3.66-3.70 (m,2H), 3.82-3.89 (m, 6H), 3.94 (dd, J=6.3, 9.5 Hz, 1H), 4.07-4.14 (m, 2H),4.96 (d, J=9.4 Hz, 1H), 4.98-5.01 (m, 1H), 5.53-5.56 (m, 1H), 5.64-5.68(m, 2H), 6.52-6.53 (m, 2H), 7.18-7.27 (m, 5H), 7.76 (d, J=9.4 Hz, 1H);¹³C NMR (125 MHz, CDCl₃): δ 24.4, 25.7, 28.3, 32.3, 32.4, 35.6, 45.2,51.9, 54.9, 55.6, 69.5, 69.9, 70.7, 71.9, 73.2, 101.9, 104.9, 109.2,119.3, 126.4, 128.0, 128.4, 129.3, 133.4, 133.9, 137.6, 155.3, 158.0,164.5; ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₂H₄₂N₂O₉S, 631.2689.found, 631.2698.

Example 39

14cZ: ¹H NMR (500 MHz, CDCl₃): δ 1.48-1.52 (m, 3H), 1.57-1.72 (m, 4H),2.10-2.14 (m, 1H), 2.28-2.32 (m, 1H), 2.47-2.51 (m, 1H), 2.78 (dd, J=9,14 Hz, 1H), 2.87-2.91 (m, 1H), 2.98-3.08 (m, 3H), 3.45-3.3.60 (m, 1H),3.65-3.71 (m, 1H), 3.80-3.90 (m, 6H), 3.95 (dd, J=6, 9.5 Hz, 1H),4.07-4.10 (m, 1H), 4.18-4.21 (m, 1H), 4.95 (d, J=9.5 Hz, 1H), 4.98-5.02(m, 1H), 5.44-5.49 (m, 2H), 5.63 (d, J=5.5 Hz, 1H), 6.51 (dd, J=2.5, 9.8Hz, 1H), 6.53 (d, J=2 Hz, 1H), 7.18-7.27 (m, 5H), 7.81 (d, J=9 Hz, 1H);¹³C NMR (125 MHz, CDCl₃): δ 24.7, 24.8, 25.7, 27.7, 35.5, 45.2, 46.9,50.2, 54.9, 55.6, 69.5, 69.9, 70.6, 70.7, 73.2, 101.9, 104.8, 109.1,120.5, 126.4, 127.6, 128.4, 129.3, 132.5, 133.2, 137.6, 155.3, 158.1,164.6; ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₂H₄₂N₂O₉S, 631.2689.found, 631.2706.

Example 40 Inhibitor 14d

Title compound was obtained from 13d and Grubbs 1^(st) gen. cat. asdescribed for 14a in 71% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 1.40-1.47(m, 1H), 1.58-1.62 (m, 2H), 1.92-1.95 (m, 1H), 2.11-2.15 (m, 1H),2.28-2.39 (m, 2H), 2.73-2.78 (m, 1H), 2.80-3.05 (m, 6H), 3.64-3.70 (m,3H), 3.80-3.89 (m, 6H), 3.93-3.96 (m, 2H), 4.12-4.15 (m, 1H), 4.62 (brs, 1H), 4.97-4.99 (m, 1H), 5.11 (d, J=9.2 Hz, 1H), 5.52-5.54 (m, 2H),5.61 (d, J=5.1 Hz, 1H), 6.46-6.49 (m, 2H), 7.18-7.26 (m, 5H), 7.80 (d,J=9.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 22.7, 25.1, 25.7, 29.6, 35.5,41.2, 45.3, 47.8, 50.2, 54.8, 55.6, 65.9, 69.5, 70.1, 70.7, 73.2, 101.0,104.3, 109.2, 118.7, 123.3, 126.4, 128.4, 129.3, 133.4, 133.6, 137.5,155.4, 157.8, 164.7; ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₁H₄₀N₂O₉S,617.2533. found, 617.2534.

Example 41 Inhibitor 14e

Title compound was obtained from 13e and Grubbs 2^(nd) gen. cat. asdescribed for 14a in 52% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 1.46-1.51(m, 1H), 1.60-1.76 (m, 4H), 1.88-1.92 (m, 1H), 2.23-2.37 (m, 2H), 2.79(dd, J=9, 14 Hz, 1H), 2.88-3.01 (m, 2H), 3.10-3.13 (m, 1H), 3.66-3.72(m, 2H), 3.72-3.89 (m, 5H), 3.92-3.99 (m, 2H), 4.07-4.15 (m, 2H), 4.94(d, J=8.5 Hz, 1H), 5.00-5.04 (m, 1H), 5.52-5.54 (m, 2H), 5.64 (d, J=5.1Hz, 1H), 6.43 (d, J=2 Hz, 1H), 6.49-6.51 (m, 1H), 7.16-7.28 (m, 5H),7.81 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 25.7, 26.3, 26.5,29.6, 35.5, 43.9, 45.2, 50.7, 54.7, 55.6, 69.5, 69.7, 70.7, 73.3, 100.0,103.9, 109.2, 117.7, 122.9, 128.4, 129.3, 133.7, 135.1, 137.4, 155.3,157.7, 164.9; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₁H₄₀N₂O₉S,639.2352. found, 639.2345.

Example 42 Inhibitor 14f

Title compound was obtained from 13f and Grubbs 2^(nd) gen. cat. asdescribed for 14a in 81% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 1.40-1.47(m, 1H), 1.58-1.62 (m, 2H), 1.92-1.95 (m, 1H), 2.11-2.15 (m, 1H),2.28-2.39 (m, 2H), 2.73-2.78 (m, 1H), 2.80-3.05 (m, 6H), 3.64-3.70 (m,3H), 3.80-3.89 (m, 6H), 3.93-3.96 (m, 2H), 4.12-4.15 (m, 1H), 4.62 (brs, 1H), 4.97-4.99 (m, 1H), 5.11 (d, J=9.2 Hz, 1H), 5.52-5.54 (m, 2H),5.61 (d, J=5.1 Hz, 1H), 6.46-6.49 (m, 2H), 7.18-7.26 (m, 5H), 7.80 (d,J=9.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 22.7, 25.1, 25.7, 29.6, 35.5,41.2, 45.3, 47.8, 50.2, 54.8, 55.6, 65.9, 69.5, 70.1, 70.7, 73.2, 101.0,104.3, 109.2, 118.7, 123.3, 126.4, 128.4, 129.3, 133.4, 133.6, 137.5,155.4, 157.8, 164.7; ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₀H₃₈N₂O₉S,603.2376. found, 603.2369.

Example 43 Inhibitor 14g

Title compound was obtained from 13g and Grubbs 2^(nd) gen. cat. asdescribed for 14a in 81% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 1.47-1.55(m, 1H), 1.64-1.70 (m, 2H), 2.51-2.54 (m, 1H), 2.73-2.78 (m, 1H),2.84-3.94 (m, 2H), 3.04-3.11 (m, 2H), 3.19 (d, J=13.6 Hz, 1H), 3.70-3.75(m, 2H), 3.86 (s, 3H), 3.94-3.99 (m, 4H), 4.07-4.08 (m, 1H), 4.18 (br s,1H), 4.58 (s, 1H), 5.02-5.06 (m, 1H), 5.12 (s, 1H), 5.61 (d, J=5 Hz,1H), 5.76-5.82 (m, 1H), 5.88-5.93 (m, 1H), 6.35 (d, J=1.1 Hz, 1H), 6.51(dd, J=1.5, 8.7 Hz, 1H), 7.20-7.31 (m, 5H), 7.79 (d, J=8.8 Hz, 1H); ¹³CNMR (125 MHz, CDCl₃): δ 25.8, 26.7, 35.4, 43.6, 45.3, 47.1, 55.1, 55.7,67.4, 69.6, 70.8, 73.3, 99.4, 104.1, 109.2, 120.5, 126.5, 126.9, 128.5,129.4, 131.2, 132.0, 137.6, 155.6, 157.4, 164.7; ESI (+) HRMS (m/z):[M+Na]⁺ calcd for C₂₉H₃₆N₂O₉S, 611.2039. found, 611.2040.

Example 44 Inhibitor 14h

Title compound was obtained from 13h and Grubbs 2^(nd) gen. cat. asdescribed for 14a in 79% yield after flash-chromatography (2:3hexanes:EtOAc) as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 1.45-1.51(m, 1H), 1.60-1.68 (m, 2H), 2.80-2.90 (m, 2H), 3.00-3.12 (m, 3H), 3.53(br s, 1H), 3.66-3.71 (m, 2H), 3.83 (s, 4H), 3.92-3.95 (m, 3H),4.88-5.01 (m, 3H), 5.13 (d, J=8.3 Hz, 1H), 6.59-6.64 (m, 2H), 7.19-7.26(m, 5H), 7.74 (d, J=8.7 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 25.7, 29.6,35.3, 43.9, 45.2, 47.8, 55.1, 55.7, 69.5, 69.7, 70.7, 71.1, 73.3, 104.5,107.2, 109.2, 123.4, 126.1, 126.4, 128.4, 129.3, 131.3, 131.5, 137.6,155.6, 157.7, 164.5; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₂₈H₃₄N₂O₉S,597.1883. found, 597.1887.

Example 45 Inhibitor 15a

To a stirring solution of 14a (10 mg, 0.015 mmol) in EtOAc (2 mL) wasadded 10% Pd on carbon and the reaction was stirred under H₂ atmospherefor 12 h. After this time the reaction was filtered through a pad ofcelite and solvent was evaporated under reduced pressure. The residuewas then purified by flash-chromatography to give 15a (9.2 mg, 93%yield) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 1.40-1.47 (m, 3H),1.49-1.52 (m, 2H), 1.71-1.83 (m, 4H), 2.75 (dd, J=9.5, 13.7 Hz, 1H),2.86-2.91 (m, 1H), 3.01-3.10 (m, 3H), 3.64-3.71 (m, 2H), 3.85 (s, 1H),3.94 (dd, J=6.4, 9.5 Hz, 1H), 4.01-4.04 (m, 1H), 4.10-4.16 (m, 1H), 4.94(d, J=9.2 Hz, 1H), 5.01-5.15 (m, 1H), 5.67 (d, J=5.1 Hz, 1H), 6.52-6.54(m, 2H), 7.19-7.29 (m, 5H), 7.85 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 22.4, 22.9, 23.0, 23.8, 24.2, 25.5, 26.3, 26.4, 28.2, 29.1,29.4, 29.9, 35.3, 45.1, 51.3, 52.8, 54.6, 55.4, 69.2, 69.3, 69.5, 70.5,71.9, 73.1, 100.2, 103.9, 109.0, 118.7, 126.3, 128.2, 129.1, 133.5,137.3, 155.1, 157.9, 164.6; ESI (+) HRMS (ink): [M+Na]⁺ calcd forC₃₄H₄₈N₂O₉S, 683.2978. found, 683.2984.

Example 46 Inhibitor 15b

Title compound was obtained from 14b as described for 15a in 90% yieldafter flash-chromatography (2:3 hexanes:EtOAc) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ 1.30-1.47 (m, 8H), 1.57-1.70 (m, 5H), 1.72-1.79 (m,1H), 1.82-1.87 (m, 2), 2.77 (dd, J=10, 14 Hz, 1H), 2.86-2.91 (m, 1H),3.00 (dd, J=4.5, 14 Hz, 1H), 3.05 (d, J=2.5, 15 Hz, 1H), 3.14-3.23 (m,2H), 3.56-3.62 (m, 2H), 3.65-3.72 (m, 2H), 3.81-3.90 (m, 6H), 3.94 (dd,J=6.5, 10 Hz, 1H), 4.05-4.15 (m, 2H), 4.96 (d, J=9.5 Hz, 1H), 4.99-5.02(m, 1H), 5.64 (d, J=5.5 Hz, 1H), 6.49-6.52 (m, 2H), 7.17-7.27 (m, 5H),7.82 (d, J=9.5 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 23.2, 24.2, 24.9,25.2, 25.7, 26.1, 27.6, 28.9, 35.4, 45.2, 49.2, 52.7, 54.8, 55.6, 68.9,69.5, 70.7, 71.7, 73.2, 100.5, 104.2, 109.2, 118.5, 126.4, 128.4, 129.3,133.8, 137.5, 155.3, 158.1, 164.8; ESI (+) HRMS (m/z): [M+Na]⁺ calcd forC₃₃H₄₆N₂O₉S, 669.2822. found, 669.2828.

Example 47 Inhibitor 15c

Title compound was obtained from 14c as described for 15a in 90% yieldafter flash-chromatography (2:3 hexanes:EtOAc) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ 1.35-1.39 (m, 3H), 1.40-1.54 (m, 5H), 1.60-1.66 (m,7H), 1.85-1.98 (m, 2), 2.78 (dd, J=9.1, 14 Hz, 1H), 2.88-2.91 (m, 1H),2.97-3.03 (m, 1H), 3.06-3.14 (m, 1H), 3.39-3.43 (m, 1H), 3.50-3.56 (m,1H), 3.66-3.72 (m, 3H), 3.82-3.85 (m, 6H), 3.95 (dd, J=6.3, 9.6 Hz, 1H),4.07-4.10 (m, 2H), 4.95-5.03 (m, 2H), 5.64 (d, J=5 Hz, 1H), 6.50-6.53(m, 2H), 7.17-7.27 (m, 5H), 7.82 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 22.6, 23.5, 23.9, 24.6, 25.3, 25.7, 26.1, 29.3, 29.6, 31.8,35.5, 44.8, 45.2, 46.5, 51.0, 54.8, 55.6, 69.5, 70.1, 70.3, 70.7, 70.8,73.2, 100.9, 104.3, 109.2, 118.5, 126.4, 128.4, 129.4, 133.9, 137.5,155.3, 158.2, 164.8; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₂H₄₄N₂O₉S,655.2668. found, 655.2667.

Example 48 Inhibitor 15d

Title compound was obtained from 14d or 14e as described for 15a in 94%yield after flash-chromatography (2:3 hexanes:EtOAc) as a white solid.¹H NMR (500 MHz, CDCl₃): δ 1.33-1.54 (m, 6H), 1.57-1.74 (m, 3H),1.86-1.94 (m, 2H), 2.77 (dd, J=9.5, 14 Hz, 1H), 2.86-2.90 (m, 1H),2.96-3.08 (m, 3H), 3.24-3.29 (m, 1H), 3.64-3.72 (m, 2H), 3.74-3.86 (m,7H), 3.94-4.00 (m, 2H), 4.15-4.22 (m, 2H), 4.92 (d, J=9.2 Hz, 1H),4.97-5.02 (m, 1H), 5.62 (d, J=5.2 Hz, 1H), 6.48 (d, J=2.1 Hz, 1H), 6.52(dd, J=2, 8.9 Hz, 1H), 7.17-7.27 (m, 5H), 7.87 (d, J=8.6 Hz, 1H); ¹³CNMR (125 MHz, CDCl₃): δ 23.2, 25.0, 25.6, 26.1, 26.3, 29.6, 35.4, 42.9,45.2, 48.5, 54.7, 55.6, 69.3, 69.5, 70.7, 73.3, 99.9, 103.8, 109.1,117.1, 126.4, 128.4, 129.3, 134.6, 137.4, 155.1, 157.7, 165.2; ESI (+)HRMS (m/z): [M+Na]⁺ calcd for C₃₁H₄₂N₂O₉S, 641.2509. found, 641.2512.

Example 49 Inhibitor 15e

Title compound was obtained from 14f as described for 15a in 93% yieldafter flash-chromatography (2:3 hexanes:EtOAc) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ 1.33-1.48 (m, 4H), 1.56-1.66 (m, 3H), 1.67-1.71 (m,2H), 1.83-1.86 (m, 1H), 2.04-2.12 (m, 2H), 2.74 (dd, J=9.7, 14 Hz, 1H),2.83-2.99 (m, 4H), 3.18 (br s, 1H), 3.64-3.71 (m, 2H), 3.76-3.85 (m,6H), 3.89-3.99 (m, 3H), 4.08 (br s, 1H), 4.19-4.23 (m, 1H), 4.96-5.00(m, 2H), 5.63 (d, J=5 Hz, 1H), 6.45 (d, J=2 Hz, 1H), 6.50 (dd, J=2.5, 9Hz, 1H), 7.15-7.26 (m, 5H), 7.88 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz,CDCl₃): δ 22.2, 24.4, 24.6, 25.7, 25.8, 35.4, 45.2, 46.8, 51.8, 54.7,55.7, 69.2, 69.6, 70.5, 70.8, 73.3, 99.8, 103.9, 109.2, 117.5, 126.5,128.4, 129.3, 137.4, 155.2, 157.8, 165.3; ESI (+) HRMS (m/z): [M+Na]⁺calcd for C₃₀H₄₀N₂O₉S, 605.2533. found, 605.2526.

Example 50 Inhibitor 15f

Title compound was obtained from 14g as described for 15a in 90% yieldafter flash-chromatography (2:3 hexanes:EtOAc) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ 1.46-1.50 (m, 3H), 1.59-1.67 (m, 4H), 1.96-2.04 (m,2H), 2.81-2.91 (m, 2H), 3.03 (dd, J=3, 14 Hz, 1H), 3.07-3.12 (m, 2H),3.83-3.91 (m, 6H), 3.93-4.02 (m, 3H), 4.25 (br s, 1H), 4.98-5.02 (m,2H), 5.63 (d, J=5 Hz, 1H), 6.40 (d, J=2 Hz, 1H), 6.49 (dd, J=2, 9 Hz,1H), 7.18-7.28 (m, 5H), 7.76 (d, J=9 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃):δ 22.4, 24.4, 25.3, 25.7, 35.4, 43.1, 45.2, 47.3, 54.9, 55.6, 69.5,70.1, 70.6, 73.2, 100.5, 104.2, 109.2, 121.4, 126.4, 128.4, 129.3,131.5, 137.5, 155.4, 157.7, 165.6; ESI (+) HRMS (m/z): [M+H]⁺ calcd forC₂₉H₃₈N₂O₉S, 591.2376. found, 591.2381.

Example 51 Inhibitor 15g

Title compound was obtained from 14h as described for 15a in 92% yieldafter flash-chromatography (2:3 hexanes:EtOAc) as a white solid. ¹H NMR(500 MHz, CDCl₃): δ 1.39-1.51 (m, 3H), 1.60-1.68 (m, 4H), 1.72-1.81 (m,2H), 2.82-2.92 (m, 2H), 3.00-3.06 (m, 3H), 3.66-3.71 (m, 3H), 3.80-3.87(m, 6H), 3.95 (dd, J=6, 9.5 Hz, 1H), 4.33-4.42 (m, 2H), 4.98-5.02 (m,2H), 5.63 (d, J=5 Hz, 1H), 6.62-6.64 (m, 2H), 7.18-7.28 (m, 5H), 7.77(d, J=10.5 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃): δ 24.4, 25.0, 25.7, 35.3,45.2, 46.5, 55.6, 69.5, 70.6, 73.2, 73.9, 105.6, 107.3, 109.1, 126.4,128.4, 129.3, 130.9, 137.3, 155.5, 157.4, 164.4; ESI (+) HRMS (m/z):[M+H]⁺ calcd for C₂₈H₃₆N₂O₉S, 577.2220. found, 577.2222.

Example 52 tert-Butyl(2S,3R)-4-(2,2-dimethylpent-4-enylamino)-3-hydroxy-1-phenylbutan-2-ylcarbamate(17a)

Under argon, combined amine 16a (1.58 g, 4.33 mmol) and epoxide 8 (304mg, 1.15 mmol) and heated to 60° C. for 4 h. Let stir overnight at rt.Purified by silica chromatography (3:97 MeOH:CH₂Cl₂) to give 370 mg(98.3% yield) of product as a clear oil that solidified uponrefrigeration. TLC 5:95 MeOH:CH₂Cl₂, R_(f)=0.2, visualized withninhydrin; mp 35-37° C. [α]_(D) ²⁰ +0.9 (c 0.14, CHCl₃); ¹H NMR (CDCl₃,400 MHz): δ 7.26 (m, 5H), 5.81 (m, 1H), 5.03 (m, 2H), 4.79 (d, J=9.2 Hz,1H), 3.82 (m, 1H), 3.45 (m, 1H), 2.98 (dd, J=4.8, 9.2 Hz, 1H), 2.87 (dd,J=8, 14.8 Hz, 1H), 2.69 (d, J=4.8 Hz, 2H), 2.35 (s, 2H), 2.01 (d, J=7.6Hz, 2H), 1.36 (s, 9H), 0.90 (s, 6H); ¹³C NMR (CDCl₃, 100 MHz): δ 155.8,137.9, 135.2, 129.4, 128.3, 126.2, 116.9, 79.1, 70.3, 60.1, 54.3, 52.1,44.5, 36.8, 34.3, 29.6, 25.4, 25.4; FTIR (NaCl) v_(max)=3349, 3064,3027, 2958, 2928, 1693, 1638, 1604, 1498, 1455, 1391, 1366, 1250, 1171,1126, 1042, 1017, 913, 742, 700 cm⁻¹; ESI (+) MS m/z (relativeintensity): 376.06 (100%).

Example 53 tert-Butyl(2S,3R)-4-(N-(2,2-dimethylpent-4-enyl)-2-(hex-5-enyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(18a)

Combined amine 17a (188.3 mg, 0.5 mmol) and sulfonyl chloride 7 (182.9mg, 0.6 mmol) into a reaction flask and cooled to 0° C. Under argon,added pyridine (8 mL, freshly distilled over KOH) and allowed thereaction to come to rt while stirring. The reaction turned orange andwas allowed to stir overnight. The reaction was condensed under reducedpressure, washed with sat. Cu₂SO₄ (10 mL), and the product extractedinto dichloromethane (3×20 mL). The organic layer was washed with H₂O(10 mL), brine (2×5 mL), dried over sodium sulfate and purified bysilica chromatography (20:80 EtOAc:Hexane) to give 179.9 mg (55.8%yield) of product as a clear oil. TLC 30:70 EtOAc:Hexane, R_(f)=0.46, UVand iodine to visualize; [α]_(D) ²⁰ +18.3 (c 0.12, CHCl₃); ¹H NMR(CDCl₃, 400 MHz): δ 7.78 (d, J=8.4 Hz, 1H), 7.2 (m, 5H), 6.47 (m, 2H),5.79 (m, 2H), 5.00 (m, 4H), 4.40 (d, J=8.8 Hz, 1H), 4.00 (m, 2H), 3.83(s, 3H), 3.80 (m, 1H), 3.66 (m, 1H), 3.20 (m, 4H), 2.86 (dd, J=4.8, 14.4Hz, 1H), 2.77 (dd, J=8, 13.2 Hz, 1H), 2.12 (q, J=6.8 Hz, 2H), 1.99 (d,J=7.2 Hz, 2H), 1.86 (p, J=8 Hz, 2H), 1.59 (m, 2H), 1.31 (s, 9H), 1.25(m, 1H), 0.92 (s, 3H), 0.90 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ 164.8,157.5, 155.5, 138.0, 137.6, 134.7, 134.0, 129.4, 128.3, 126.2, 118.5,117.5, 115.1, 104.2, 100.1, 79.3, 72.0, 69.1, 62.4, 55.6, 55.5, 54.2,45.2, 35.8, 33.2, 28.4, 28.2, 28.0, 25.6, 25.5, 25.0; FTIR (NaCl)v_(max)=3412, 2924, 2852, 1701, 1596, 1496, 1456, 1391, 1367, 1326,1254, 1206, 1158, 1074, 1030, 914 cm⁻¹; ESI (+) LRMS m/z (relativeintensity): 667.12 (100%); ESI (+) HRMS (m/z): [M+Na⁺] calcd forC₃₅H₅₂N₂O₇S 667.3393. found, 667.3399.

Example 54 (3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl(2S,3R)-4-(N-(2,2-dimethylpent-4-enyl)-2-(hex-5-enyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(19a)

Dissolved 18a (180 mg, 0.28 mmol) into CH₂Cl₂ (5 mL) and cooled to 0° C.Trifluoroacetic acid (1.5 mL) was added dropwise while stiffing. Thereaction was allowed to warm to rt and stir overnight. Solvents wereremoved under reduced pressure. The residue was made basic with amixture of sat. NaHCO₃ (1 mL) and 1N NaOH (1 mL) and extracted withether (5×5 mL). Solvents were removed under reduced pressure to affordthe crude amine product. This amine (0.28 mmol) was dissolved into MeCN(20 mL) and mixed with carbonate 12 (92 mg, 0.31 mmol) under argon.DIPEA (1 mL) and pyridine (1 mL) were was added dropwise and thereaction was allowed to stir overnight at rt. After stiffing for 2 days,the solvents were removed under reduced pressure and the crude materialpurified by silica chromatography (50:50 EtOAc:Hexane) to give 143.5 mg(73.1% yield over two steps) of product as a white tacky solid. TLC50:50 EtOAc:Hexane, R_(t)=0.27, UV and iodine visualization; m.p.=49-52°C.; [α]_(D) ²⁰ +3.9 (c 1.03, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.79 (d,J=8.4 Hz, 1H), 7.18 (m, 5H), 6.48 (m, 2H), 5.77 (m, 2H), 5.60 (d, J=5.2Hz, 1H), 5.02 (m, 4H), 4.87 (d, J=9.6 Hz, 1H), 4.03 (m, 2H), 3.91 (m,3H), 3.83 (s, 3H), 3.78 (m, 2H), 3.63 (m, 2H), 3.29 (m, 2H), 3.08 (m,2H), 2.94 (dd, J=4, 14.4 Hz, 1H), 2.84 (m, 1H), 2.69 (dd, J=9.6, 14 Hz),2.11 (m, 2H), 2.02 (m, 3H), 1.86 (m, 2H), 1.58 (m, 3H), 1.35 (m, 1H),0.91 (s, 3H), 0.90 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 164.9, 157.6,155.0, 137.9, 137.5, 134.5, 134.0, 129.2, 128.3, 126.3, 118.0, 117.6,115.0, 109.2, 104.3, 100.1, 73.1, 72.3, 70.6, 69.5, 69.2, 62.6, 55.6,55.6, 54.8, 45.2, 45.1, 35.7, 35.5, 33.2, 28.3, 25.7, 25.5, 25.4, 24.9;FTIR (NaCl) v_(max)=701.6, 767.6, 919.1, 1021.0, 1074.3, 1140.9, 1206.5,1256.3, 1323.6, 1595.4, 1722.9, 2922.7, 2963.6, 3076.2, 3487.2 cm⁻¹; ESI(+) MS m/z (relative intensity): 1422.15 (5%), 700.82 (100%), 588.84(26%), 545.10 (44%); ESI (+) HRMS (m/z): [M+H]⁺ calcd for C₃₇H₅₂N₂O₉S701.3472. found, 701.3473.

Example 55 Inhibitors 20a and 21a

19a (100 mg, 0.143 mmol) was dissolved into CH₂Cl₂ (90 mL). Grubbs'2^(nd) gen. cat. (11.8 mg, 0.014 mmol) was added and the reaction wasallowed to stir overnight at rt. Solvent was removed under reducedpressure and the material purified by silica chromatography (50:50→75:25EtOAc:Hexane) to give 92.1 mg (95.7% yield) of product as a mixture ofstereoisomers (31:69 Z:E by HPLC) as a white solid. The individualstereoisomers were isolated by reversed-phase HPLC YMC-Pack ODSA (250×10mm, 5 micron); flow rate=1.5 mL/min; isocratic 80:20 MeOH:H₂O; T=25° C.;λ=210 nm, R_(t) Z=17 min, R_(t) E=18 min). 21a: [α]_(D) ²⁰ −0.8 (c 2.36,CHCl₃); ¹H NMR (CDCl₃, 800 MHz) δ 7.79 (d, J=8.8 Hz, 1H), 7.24 (t, J=8Hz, 2H), 7.18 (t, J=7.2 Hz, 1H), 7.12 (d, J=7.2 Hz, 2H), 6.50 (dd,J=1.6, 8.8 Hz, 1H), 6.47 (m, 1H), 5.68 (m, 1H), 5.62 (d, J=5.6 Hz, 1H),5.60 (d, J=1H), 4.98 (q, J=7.2 Hz, 1H), 4.71 (d, J=9.6 Hz, 1H),4.04-3.88 (m, 4H), 3.84 (s, 3H), 3.82 (m, 1H), 3.77 (m, 1H), 3.71 (dd,J=6.4, 13.6 Hz, 1H), 3.68 (m, 1H), 3.64 (m, 1H), 3.11 (dd, J=9.6, 15.2Hz, 1H), 2.97 (dd, J=2.4, 15.2 Hz, 1H), 2.92 (dd, J=4, 14.4 Hz, 1H),2.86 (q, J=7.2 Hz, 1H), 2.68 (dd, J=9.6, 14.4 Hz, 1H), 2.10-1.94 (m,4H), 1.90-1.72 (m, 4H), 1.58 (m, 1H), 1.39 (m, 1H), 1.25 (s, 1H), 1.24(t, J=6.4 Hz, 1H), 1.12 (s, 3H), 1.05 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz)δ 164.9, 158.1, 155.1, 137.5, 134.5, 133.6, 129.3, 128.5, 128.4, 126.5,117.8, 109.3, 104.4, 100.6, 73.3, 72.2, 70.7, 69.7, 69.6, 63.0, 56.1,55.7, 54.7, 46.1, 45.3, 35.8, 35.6, 29.7, 29.6, 29.2, 27.6, 26.1, 25.7;FTIR (film, NaCl) v_(max)=3445, 3343, 2926, 1720, 1596, 1575, 1532,1494, 1469, 1445, 1429, 1390, 1369, 1325, 1256, 1207, 1172, 1141, 1074,1021, 988, 964, 926, 896, 841, 753, 702, 665, 643 cm⁻¹; ESI (+) LRMS m/z(relative intensity): 695.30 (70%), 1366.74 (100%); ESI (+) HRMS (m/z):[M+Na]⁺ calcd for C₃₅H₄₈N₂O₉S 695.2978. found, 695.2989.

Example 56

20a: [α]_(n) ²⁰ −0.5 (c 0.99, CHCl₃); ¹H NMR (CDCl₃, 800 MHz) δ 7.81 (d,J=8.8 Hz, 1H), 7.23 (t, J=8 Hz, 2H), 7.18 (d, J=7.2 Hz, 1H), 7.13 (d,J=7.2 Hz, 2H), 6.51 (dd, J=2.4, 8.8 Hz, 1H), 6.47 (d, J=2.4 Hz, 1H),5.65 (q, J=8.8 Hz, 1H), 5.62 (d, J=5.6 Hz, 1H), 5.53 (q, J=8.8 Hz, 1H),4.98 (q, J=5.6 Hz, 1H), 4.74 (d, J=9.6 Hz, 1H), 4.08 (t, J=4.8 Hz, 2H),3.96 (m, 1H), 3.93 (dd, J=6.4, 9.6 Hz, 1H), 3.85 (s, 3H), 3.82 (dt,J=2.4, 8.8 Hz, 1H), 3.79 (m, 1H), 3.68 (dd, J=6.4, 9.6 Hz, 1H), 3.65 (m,1H), 3.08 (dd, J=8.8, 15.2 Hz, 1H), 2.93 (dd, J=4, 14.4 Hz, 1H), 2.91(dd, J=0.8, 15.2 Hz, 1H), 2.86 (m, 1H), 2.70 (dd, J=9.6, 14.4 Hz, 1H),2.20-1.98 (m, 4H), 1.90 (br s, 2H), 1.76-1.62 (m, 2H), 1.57 (m, 1H),1.38 (m, 1H), 1.25 (s, 3H), 1.13 (s, 3H), 1.07 (s, 3H); ¹³C NMR (CDCl₃,125 MHz) δ 164.9, 157.9, 155.1, 137.5, 134.8, 131.0, 129.3, 128.4,126.7, 126.5, 117.3, 109.3, 104.1, 100.2, 73.3, 72.6, 70.7, 69.6, 68.8,64.1, 56.1, 55.7, 54.7, 45.3, 40.5, 36.1, 35.6, 29.7, 27.4, 26.7, 26.1,25.8, 25.3; FTIR (film, NaCl) v_(max)=3344, 2925, 1718, 1595, 1575,1534, 1493, 1445, 1388, 1325, 1257, 1206, 1171, 1141, 1074, 1021, 925,892, 933, 756, 702 cm⁻¹; ESI (+) LRMS m/z (relative intensity): 695.28(95%), 1366.73 (100%); ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₅H₄₈N₂O₉S695.2978. found, 695.2970.

Example 57

22a: The corresponding olefin 20a or 21a (21.6 mg, 0.032 mmol) wasdissolved into ethyl acetate (5 mL). Pd/C (10%) was added and thereaction flask evacuated with H₂ gas. After stirring overnight, thereaction was filtered over celite and purified by silica chromatography(50:50→75:25 EtOAc:Hexane) to give 19.49 mg (90.2% yield) of the desiredproduct. TLC 70:30 EtOAc:Hexane, R_(t)=0.63, UV and/or phosphomolybdicacid used to visualize; [α]_(D) ²⁰ −2.9 (c 0.70, CHCl₃); ¹H NMR (CDCl₃,800 MHz) δ 7.82 (d, J=8.8 Hz, 1H), 7.24 (t, J=8 Hz, 2H), 7.18 (t, J=7.2Hz, 1H), 7.15 (d, J=7.2 Hz, 2H), 6.51 (m, 2H), 5.63 (d, J=4.8 Hz, 1H),4.98 (q, J=5.6 Hz, 1H), 4.75 (d, J=9.6 Hz, 1H), 4.10 (m, 1H), 4.07 (m,1H), 3.94 (m, 1H), 3.93 (dd, J=6.4, 9.6 Hz, 1H), 3.86 (s, 3H), 3.82 (dt,J=1.6, 8 Hz, 1H), 3.80 (m, 1H), 3.70-3.63 (m, 2H), 3.19 (dd, J=9.6, 15.2Hz, 1H), 3.01 (dd, J=1.6, 15.2 Hz, 1H), 2.98 (dd, J=4, 14.4 Hz, 1H),2.87 (m, 1H), 2.71 (dd, J=9.6, 14.4 Hz, 1H), 1.81 (m, 2H), 1.69 (m, 1H),1.64-1.54 (m, 3H), 1.45 (m, 4H), 1.38 (m, 3H), 1.27 (m, 2H), 1.18 (m,1H), 1.03 (s, 3H), 0.97 (s, 3H); ¹³C NMR (CDCl₃, 125 MHz) δ 164.9,158.1, 155.1, 137.5, 135.6, 129.3, 128.4, 126.5, 117.7, 109.3, 104.0,100.2, 73.3, 72.5, 70.7, 69.6, 68.9, 61.8, 56.1, 55.7, 54.8, 45.3, 39.8,35.8, 35.7, 28.3, 27.4, 26.2, 25.8, 25.4, 25.0, 22.3, 19.6; FTIR (NaCl)v_(max)=3449, 3333, 2930, 1718, 1596, 1534, 1444, 1325, 1257, 1206,1140, 1074, 1020, 755, 701 cm⁻¹; ESI (+) LRMS m/z (relative intensity):697.10 (27%), 674.81 (100%); ESI (+) HRMS (m/z): [M+H]⁺ calcd forC₃₅H₅₀N₂O₉S 675.3315. found, 675.3318.

Example 58 tert-Butyl(2S,3R)-3-hydroxy-4-(2-methylpent-4-enylamino)-1-phenylbutan-2-ylcarbamate(17b and 17c)

2-methylpent-4-en-1-amine (4.6 mmol) and epoxide 8 (361.5 mg, 1.3 mmol)were weighed into a flask. The flask was evacuated with argon and thereaction allowed to stir at 60° C. overnight. The crude material waspurified by silica chromatography (3:97 MeOH:CH₂Cl₂) to give 470 mg(94.6%) of product as a white solid as a mix of diastereomers (50:50 byNMR). TLC 10:90 MeOH:CH₂Cl₂ R_(f)=0.1 visualized with ninhydrin stain;mp 89-90° C.; [α]_(D) ²⁰ +5.4 (c 2.58, CHCl₃); ¹H NMR (CDCl₃, 400 MHz):δ 7.29 (m, 4H), 7.22 (m, 6H), 5.77 (m, 2H), 5.04 (m, 4H), 4.77-4.55 (m,2H), 3.85-3.59 (m, 4H), 3.45 (m, 2H), 3.01-2.6 (m, 10H), 2.52 (dd, J=6,12 Hz, 2H), 2.40 (dd, J=6.8, 11.6 Hz, 2H), 2.13 (m, 2H), 1.92 (m, 2H),1.67 (m, 2H), 1.35 (s, 18H), 0.92 (d, J=2.4, 3H), 0.91 (d, J=2 Hz, 3H;¹³C NMR (CDCl₃, 100 MHz): δ 155.9, 137.9, 137.0, 129.5, 128.4, 126.3,116.0, 79.3, 70.6, 55.7, 54.1, 51.4, 51.4, 39.2, 39.2, 36.7, 33.2, 28.3,17.8; FTIR (NaCl) v_(max)=3365, 2976, 2925, 1683, 1520, 1455, 1391,1367, 1252, 1171, 1017, 912, 755, 701 cm⁻¹; ESI (+) LRMS m/z (relativeintensity): 363.03 (100%). ESI (−) LRMS m/z (relative intensity): 361.28(100%). Pure 17c was prepared in a similar fashion by using enantiopure(S)-2-methylpent-4-en-1-amine. ¹H NMR (CDCl₃, 400 MHz): 8

Example 59 tert-Butyl(2S,3R)-4-(2-(hex-5-enyloxy)-4-methoxy-N-(2-methylpent-4-enyl)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(18band 18c)

Under argon, combined amines 17b and 17c (165.6 mg, 0.46 mmol) withsulfonyl chloride 7 (167 mg, 0.55 mmol) and cooled to 0° C. Pyridine (5mL) was added and the solution stirred overnight. Solvents were removedby reduced pressure and the crude material purified by silicachromatography (20:80 EtOAc:Hexane) to give 148 mg (51% yield) ofproduct as a clear oil. TLC 40:60 EtOAc:Hexane R_(f)=0.58 visualizedwith UV/iodine; [α]_(D) ²⁰ −1.8 (c 0.67, CHCl₃); ¹H NMR (CDCl₃, 300MHz): δ 7.83 (d, J=8.7 Hz, 2H), 7.30-7.16 (m, 10H), 6.54-6.44 (m, 4H),5.87-5.58 (m, 4H), 5.06-4.90 (m, 8H), 4.59 (m, 2H), 4.06-3.94 (m, 5H),3.84 (s, 6H), 3.73 (br, 4H), 3.36-2.80 (m, 12H), 2.26-2.06 (m, 6H),1.92-1.70 (m, 9H), 1.65-1.52 (m, 4H), 1.33 (s, 18H), 0.88 (d, J=6 Hz,3H), 0.80 (d, J=6 Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz): δ 164.7, 157.6,155.9, 138.1, 137.9, 137.7, 136.3, 133.7, 129.5, 128.3, 126.3, 119.0,116.3, 115.1, 104.1, 100.2, 79.4, 72.7, 72.3, 69.1, 57.1, 56.8, 55.6,54.6, 54.4, 53.3, 53.2, 38.7, 35.3, 33.2, 31.9, 31.7, 28.3, 28.2, 25.0,17.1; FTIR (NaCl) v_(max)=3392, 2929, 1702, 1640, 1596, 1577, 1495,1445, 1391, 1366, 1326, 1254, 1206, 1171, 1074, 995, 913, 837, 774, 701cm⁻¹; ESI (+) LRMS m/z (relative intensity): 630.68 (100%); ESI (+) HRMS(m/z): [M+H]⁺ calcd for C₃₄H₅₀N₂O₇S 631.3417. found, 631.3408. Pure 17cwas prepared in a similar fashion using pure 18c. ¹H NMR (CDCl₃, 400MHz): δ 7.84 (d, 0.1=8.4 Hz, 1H), 7.27 (m, 2H), 7.20 (m, 3H), 6.50 (dd,J=2.4, 8.8 Hz, 1H), 6.46 (d, J=2.4 Hz, 1H), 5.80 (m, 1H), 5.66 (m, 1H),5.05-4.93 (m, 4H), 4.57 (d, J=7.2 Hz, 1H), 4.03 (t, J=6.8 Hz, 2H), 3.96(m, 1H), 3.85 (s, 3H), 3.74 (m, 2H), 3.25 (m, 3H), 2.94 (m, 3H), 2.19(m, 1H), 2.12 (q, J=7.2 Hz, 2H), 1.87 (m, 2H), 1.74 (m, 2H), 1.59 (m,2H), 1.34 (s, 9H), 0.80 (d, J=6 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ164.7, 157.6, 155.9, 138.1, 137.7, 136.4, 133.7, 129.6, 128.4, 126.3,119.1, 116.3, 115.1, 104.1, 100.2, 79.5, 72.3, 69.1, 56.9, 55.7, 54.5,53.2, 38.1, 35.2, 33.3, 31.8, 28.4, 28.2, 25.0, 17.1.

Example 60 (3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl(2S,3R)-4-(2-(hex-5-enyloxy)-4-methoxy-N-(2-methylpent-4-enyl)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate(19b and 19c)

Dissolved 18b and 18c (140 mg, 0.22 mmol) into CH₂Cl₂ (4.5 mL) and addedTFA (1.5 mL). Stirred for 4 hr and quenched with sat. NaHCO₃ (1.5 mL).2N NaOH was added until the solution turned basic. Extracted with ether,washed with brine, and dried over Na_(s)SO₄. Solvents were removed underreduced pressure to give crudeN-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyl)-2-(hex-5-enyloxy)-4-methoxy-N-(2-methylpent-4-enyl)benzensulfonamide.Under argon, the crude amine was dissolved into MeCN (5 mL).(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl 4-nitrophenyl carbonate(71.4 mg, 0.24 mmoL) and DIPEA (0.75 mL) were added and the reactionstirred overnight. Solvents were removed under reduced pressure and thecrude material purified by silica chromatography (50:50 EtOAc:Hexane) togive 67.7 mg (45% yield) of product as a clear oil. TLC 50:50EtOAc:Hexane R_(t)=0.27 visualized by UV and iodine; [α]_(D) ²⁰ −5.7 (c0.17, CHCl₃); ¹H NMR (CDCl₃, 400 MHz): δ 7.83 (d, J=8.8 Hz, 2H), 7.24(m, 4H), 7.18 (m, 6H), 6.50 (m, 4H), 5.85-5.60 (m, 6H), 5.05-4.92 (m,12H), 4.06-3.62 (m, 24H), 3.40-2.70 (m, 14H), 2.24-1.40 (m, 22H), 0.90(d, J=6.4 Hz, 3H), 0.80 (d, J=6.4 Hz); ¹C NMR (CDCl₃, 100 MHz): δ 164.8,157.6, 155.3, 138.0, 137.6, 137.5, 136.2, 133.8, 129.3, 128.4, 126.5,118.8, 118.7, 116.5, 115.1, 109.2, 104.2, 100.3, 73.3, 72.8, 72.3, 70.7,69.6, 69.2, 57.3, 56.9, 55.7, 55.0, 54.9, 53.4, 53.2, 45.3, 38.7, 38.1,35.4, 35.4, 33.2, 32.1, 31.9, 28.4, 25.7, 25.0, 17.1; FTIR (film, NaCl)v_(max)=3436, 2970, 2927, 1718, 1600, 1458, 1374, 1106 cm⁻¹; ESI (+)LRMS m/z (relative intensity): 709.24 (100%); ESI (+) HRMS (m/z):[M+Na]⁺ calcd for C₃₆H₅₀N₂O₉S 709.3135. found, 709.3131. Pure 19c wasprepared in a similar fashion using pure 18c. ¹H NMR (CDCl₃, 400 MHz) δ7.84 (d, J=8.8 Hz, 1H), 7.26 (m, 2H), 7.20 (m, 3H), 6.52 (dd, J=2, 8.8Hz, 1H), 6.48 (d, J=2.4 Hz, 1H), 5.85-5.62 (m, 3H), 5.05-4.85 (m, 6H),4.05 (t, J=6.8 Hz, 2H), 3.95 (dd, J=6.4, 9.6 Hz, 1H), 3.89-3.77 (6H),3.75-3.65 (m, 3H), 3.38 (dd, J=9.2, 15.6 Hz, 1H), 3.24 (dd, J=8, 14 Hz,1H), 3.15 (dd, J=2.4, 15.2 Hz, 1H), 2.93 (m, 3H), 2.80 (dd, J=9.2, 14Hz, 1H), 2.23 (m, 1H), 2.12 (m, 2H), 1.90-1.45 (m, 8H), 0.81 (d, J=6 Hz,3H).

Example 61 Inhibitors 20b, 20c, 21b, and 21c

Under argon, dissolved carbamates 19b and 19c (118.5 mg, 0.17 mmol) intoCH₂Cl₂ (125 mL) and added Grubb's 2^(nd) gen. cat. (14.6 mg, 0.02 mmol).Stirred overnight. Solvents were removed under reduced pressure and thecrude material purified by silica chromatography (50:50 EtOAc:Hexane) togive 110 mg (97% yield) of product as a dirty oil. TLC 70:30EtOAc:Hexane R_(f)=0.45. Reversed-phase HPLC (Waters Sunfire C₁₈ 50×4.6mm, 5 micron coupled to Agilent Eclipse XDB C₁₈ 150×4.6 mm, 5 micron andYMC-Pack C₈ 250×4.6 mm, 5 micron, Flow rate=0.95 mL/min, X=215 nm, T=30°C., isocratic 60:40 MeCN:H₂O) was used to isolate the individualisomers: R_(t) (R Z)=22 min, R_(t) (S Z)=24 min, R_(t) (R E)=25.3 min,R_(t) (S E)=26.8 min.

Example 62

20b: [α]_(D) ²⁰ −29 (c 0.60, CHCl₃); ¹H NMR (CDCl₃, 800 MHz): δ 7.86 (d,J=8.8 Hz, 1H), 7.24 (m, 2H), 7.17 (m, 3H), 6.50 (dd, J=2.4, 8.8 Hz, 1H),6.48 (d, J=1.6 Hz, 1H), 5.63 (d, J=4.8 Hz, 1H), 5.57 (q, J=7.2 Hz, 1H),5.44 (q, J=9.6 Hz, 1H), 4.99 (q, J=6.4 Hz, 1H), 4.84 (d, J=9.6 Hz, 1H),4.25 (m, 1H), 4.11 (m, 2H), 3.93 (dd, J=6.4, 9.6 Hz, 1H), 3.85 (s, 3H),3.85-3.79 (m, 2H), 3.75-3.65 (m, 5H), 3.16 (dd, J=9.6, 15.2 Hz, 1H),2.99 (dt, J=4, 13.6 Hz, 2H), 2.87 (m, 1H), 2.81 (d, J=15.2 Hz, 1H), 2.74(dd, J=9.6, 14.4 Hz, 1H), 2.18 (m, 1H), 2.11-2.01 (m, 4H), 1.95 (m, 1H),1.86 (m, 1H), 1.73 (m, 2H), 1.62-1.50 (m, 2H), 1.12 (d, J=6.4 Hz, 3H);¹³C NMR (CDCl₃, 125 MHz): δ 137.5, 134.4, 131.1, 129.4, 128.4, 127.2,126.5, 118.1, 109.3, 103.9, 100.3, 73.3, 73.0, 70.7, 69.6, 68.4, 59.1,55.7, 54.7, 53.9, 45.3, 35.7, 32.8, 32.2, 29.7, 27.5, 26.1, 25.8, 25.4,17.4; FTIR (film, NaCl) v_(max)=3467, 3333, 2923, 1717, 1595, 1575,1533, 1495, 1444, 1384, 1324, 1256, 1206, 1139, 1073, 1021, 932, 755,702, 668 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺ calcd for C₃₆H₅₀N₂O₉S709.3135. found, 709.3131.

Example 63

20c: [α]_(D) ²⁰ −30.6 (c 0.83, CHCl₃); ¹H NMR (CDCl₃, 800 MHz): δ 7.83(d, J=8.8 Hz, 1H), 7.25 (m, 2H), 7.17 (m, 3H), 6.50 (dd, J=1.6, 8.8 Hz,1H), 6.78 (d, J=2.4 Hz, 1H), 5.66 (m, 1H), 5.64 (d, J=4.8 Hz, 1H), 5.52(m, 1H), 5.00 (q, J=5.6 Hz, 1H), 4.84 (d, J=8.8 Hz, 1H), 4.02-3.88 (m,4H), 3.86-3.78 (m, 6H), 3.74-3.65 (m, 3H), 3.11 (dd, J=8, 14.4 Hz, 1H),2.96 (dd, J=3.2, 13.6 Hz, 1H), 2.89 (m, 2H), 2.74 (dd, J=8.8, 13.6 Hz,1H), 2.18 (m, 1H), 2.09-1.94 (m, 5H), 1.90-1.78 (m, 4H), 1.69 (m, 1H),1.61 (m, 1H), 1.13 (d, J=7.2 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 164.8,158.1, 155.3, 137.6, 134.0, 133.3, 129.3, 128.5, 128.1, 126.5, 118.8,109.3, 104.3, 100.7, 73.4, 72.6, 70.8, 69.6, 58.5, 58.1, 55.7, 54.8,54.4, 45.3, 37.9, 35.4, 32.3, 30.0, 29.7, 28.6, 26.2, 25.8, 18.4, 17.7;FTIR (NaCl) v_(max)=3442, 3339, 2924, 2854, 1717, 1595, 1575, 1533,1495, 1444, 1429, 1387, 1325, 1256, 1206, 1171, 1139, 1073, 1019, 988,939, 835, 755, 701, 667 cm⁻¹; ESI (+) LRMS m/z (relative intensity):1338.65 (7%), 681.34 (100%), 659.09 (19%), 304.05 (19%); ESI (+) HRMS(m/z): [M+Na]⁺ calcd for C₃₄H₄₆N₂O₉S 681.2822. found, 681.2825.

Example 64

21b: [α]_(D) ²⁰ 27.6 (c 0.36, CHCl₃); ¹H NMR (CDCl₃, 800 MHz): δ 7.83(d, J=8.8 Hz, 1H), 7.27 (m, 2H), 7.20 (m, 3H), 6.49 (m, 2H), 5.64 (d,J=5.6 Hz, 1H), 5.57 (q, J=7.2 Hz, 1H), 5.47 (q, J=8.8 Hz, 1H), 5.00 (q,J=8 Hz, 1H), 4.88 (d, J=8.8 Hz, 1H), 4.15 (m, 1H), 4.12 (m, 1H), 3.96(m, 2H), 3.90-3.84 (m, 5H), 3.71-3.64 (m, 3H), 3.46 (m, 1H), 3.17 (m,2H), 3.12 (dd, J=4, 14.4 Hz, 1H), 3.02 (dd, J=4.8, 14.4 Hz, 1H), 2.90(m, 2H), 2.78 (dd, J=10.4, 14.4 Hz, 1H), 2.18 (m, 1H), 2.12-1.94 (m,4H), 1.86 (m, 2H), 1.72 (m, 1H), 1.60-1.50 (m, 2H), 1.09 (d, J=6.4 Hz,3H); ¹³C NMR (CDCl₃, 125 MHz): δ 164.8, 157.8, 155.6, 137.7, 134.3,130.8, 129.3, 128.5, 127.6, 126.5, 118.3, 109.3, 104.0, 100.2, 73.4,72.3, 709, 69.6, 68.4, 58.0, 55.7, 55.1, 53.3, 45.3, 35.4, 32.7, 32.2,27.3, 26.0, 25.8, 25.3, 18.0; FTIR (film, NaCl) v_(max)=3467, 3337,2927, 1717, 1595, 1575, 1538, 1495, 1456, 1325, 1255, 1206, 1141, 1073,1021, 835, 754, 702, 668 cm⁻¹; ESI (+) HRMS (m/z): [M+Na]⁺ calcd forC₃₆H₅₀N₂O₉S 709.3135. found, 709.3131.

Example 65

21c: [α]_(D) ²⁰ +20.5 (c 1.17, CHCl₃); ¹H NMR (CDCl₃, 800 MHz): δ 7.83(d, J=8.8 Hz, 1H), 7.24 (t, J=7.2 Hz, 2H), 7.18 (m, 1H), 7.14 (d, J=7.2Hz, 2H), 6.50 (dd, J=2.4, 8.8 Hz, 1H), 6.47 (d, J=2.4 Hz, 1H), 5.67 (m,1H), 5.64 (d, J=4.8 Hz, 1H), 5.53 (m, 1H), 5.00 (q, J=8 Hz, 1H), 4.76(d, J=9.6 Hz, 1H), 4.17 (br, 1H), 4.11 (m, 1H), 3.94 (dd, J=6.4, 9.6 Hz,1H), 3.88 (t, J=8.8 Hz, 1H), 3.85 (m, 4H), 3.80 (m, 1H), 3.75 (m, 1H),3.69 (m, 2H), 3.55 (m, 1H), 2.98 (m, 2H), 2.91 (m, 2H), 2.76 (dd, J=8.8,14.4 Hz, 1H), 2.47 (br, 1H), 2.26 (m, 1H), 2.17 (m, 2H), 1.93 (m, 1H),1.87 (m, 1H), 1.82-1.70 (m, 4H), 1.63 (m, 1H), 1.47 (m, 1H), 1.06 (d,J=6.4 Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz): δ 164.8, 158.1, 155.2, 137.4,134.2, 132.5, 129.6, 129.4, 128.5, 126.5, 119.0, 109.3, 104.2, 100.5,73.4, 71.5, 70.8, 69.6, 58.6, 55.7, 54.9, 54.6, 45.3, 38.2, 35.7, 33.6,30.3, 29.8, 28.1, 26.2, 25.8, 19.0; FTIR (film, NaCl) v_(max)=3463,3339, 2925, 1717, 1595, 1575, 1538, 1495, 1444, 1387, 1326, 1256, 1207,1171, 1140, 1073, 1019, 987, 920, 836, 734, 701 cm⁻¹; ESI (+) LRMS m/z(relative intensity): 1338.70 (36%), 681.25 (100%); ESI(+) HRMS (m/z):[M+Na]⁺calcd for C₃₄H₄₆N₂O₉S 681.2822. found, 681.2812.

EXAMPLE Cells and Viruses

Human CD4⁺ MT-2 and MT-4 cell lines were grown in RPMI 1640-basedculture medium supplemented with 10% fetal calf serum (PAA LaboratoriesGmbH, Linz, Austria) plus 50 U of penicillin and 100 μg of kanamycin perml. The following HIV-1 strains were used for the drug susceptibilityassay: HIV-1_(LAI), HIV-1_(NL4-3), HIV-2_(EHO), and HIV-2_(ROD), as wellas clinical HIV-1 strains from drug-naive patients with AIDS(HIV-1_(ERS104pre)) (Clavel et al. 1986 Science 233:343-6; Shirasaka etal. 1993 Proc Natl Acad Sci USA 90:562-6; Yoshimura et al. 2002 J Virol76:1349-58.), and six HIV-1 clinical isolates that were originallyisolated from patients with AIDS who had received anti-HIV-1 therapyheavily (for 32 to 83 months) and that were genotypically andphenotypically characterized as multiple-PI resistant HIV-1 variants(Yoshimura et al. 1999 Proc Natl Acad Sci USA 96:8675-80). To determinewhether each clinical HIV-1 isolate used in the present study was asyncytium-inducing (SI virus) or non-syncytium-inducing (NSI virus)strain, MT-2 cells (10⁵) were exposed to an aliquot of viral stocksupernatant containing 100 50% tissue culture infectious doses(TCID_(50S)) of the virus and cultured in 6-well culture plates.Cultures were maintained for 4 weeks and were examined under an invertedmicroscope to determine the syncytium-inducing or non-syncytium-inducingnature of the virus, as described previously (Id.).

EXAMPLE Antiviral Agents

Saquinavir (SQV) and ritonavir (RTV) were obtained from Roche Products,Ltd. (Welwyn Garden City, United Kingdom) and Abbott Laboratories(Abbott Park, Ill.), respectively. Amprenavir (APV) was obtained fromGlaxoSmithKline (Research Triangle Park, N.C.). Nelfinavir (NFV) andlopinavir (LPV) were obtained from Japan Energy, Inc., Tokyo, Japan.Indinavir (IDV) was obtained from Merck Research Laboratories (Rahway,N.J.). Atazanavir (AZV) was obtained from Bristol Myers Squibb (NewYork, N.Y.). Darunavir (DRV) was synthesized as previously described(Ghosh et al. 2004. J Org Chem 69:7822-9.). Tipranavir (TPV) wasobtained through the AIDS Research and Reference Reagent Program,Division of AIDS, NIAID, National Institutes of Health.

EXAMPLE Drug Susceptibility Assay

The susceptibility of HIV-1_(LAI) and primary HIV-1 isolates to variousdrugs were determined as described previously (Koh et al. 2003Antimicrob Agents Chemother 47:3123-9). Briefly, MT-2 cells (2×10⁴/ml)were exposed to 100 50% tissue culture infectious doses (TCID_(50S)) ofHIV-1_(LAI), in the presence or absence of various concentrations ofdrugs in 96-well microculture plates and were incubated at 37° C. for 7days. After 100 μl of the medium was removed from each well,3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)solution (10 μl, 7.5 mg/ml in phosphate-buffered saline) was added toeach well in the plate, followed by incubation at 37° C. for 3 h. Afterincubation to dissolve the formazan crystals, 100 μl of acidifiedisopropanol containing 4% (vol/vol) Triton X-100 was added to each welland the optical density was measured using a kinetic microplate reader(Vmax; Molecular Devices, Sunnyvale, Calif.). All assays were performedin duplicate or triplicate. To determine the susceptibility of primaryHIV-1 isolates to drugs, phytohemagglutinin-activated peripheral bloodmononuclear cells (PHA-PBMCs; 10₆/ml) were exposed to 50 TCID_(50S) ofeach. The target cells were exposed to HIV-1_(104pre) or drug-resistantHIV-1 in the presence or absence of various concentrations of drugs andwere incubated for 7 days. Upon the conclusion of the culture, theamounts of p24 Gag protein in the supernatants were determined using afully automated chemiluminescent enzyme immunoassay system (Lumipulse F;Fujirebio Inc., Tokyo, Japan) (Maeda et al. 2001 J Biol Chem276:35194-200). To determine the drug susceptibility of certainlaboratory HIV-1 strains, MT-4 cells were used as target cells. Inbrief, MT-4 cells (10⁵/ml) were exposed to 100 TCID_(50S) ofdrug-resistant HIV-1 strains in the presence or absence of variousconcentrations of drugs and on day 7 of culture, the supernatant washarvested and the amounts of p24 Gag protein were determined. The drugconcentrations that suppressed the production of p24 Gag protein by 50%(50% effective concentrations [EC_(50S)]) were determined by comparisonwith the level of p24 production in drug-free control cell cultures. Allassays were performed in duplicate or triplicate.

EXAMPLE Generation of PI-Resistant HIV-1 In Vitro

In the experiments for selecting drug-resistant variants, MT-4 cellswere exploited as target cells, since HIV-1 in general replicates atgreater levels in MT-4 cells than in MT-2 cells. MT-4 cells (10⁵/ml)were exposed to HIV-1NL4-3 (500 TCID50s) and cultured in the presence ofvarious PIs, each at an initial concentration of its EC50 value. Viralreplication was monitored by determining the amount of p24 Gag producedby MT-4 cells. The culture supernatants were harvested on day 7 and usedto infect fresh MT-4 cells for the next round of culture in the presenceof increasing concentrations of each drug. When the virus began topropagate in the presence of the drug, the drug concentration wasgenerally increased two- to three-fold. Proviral DNA samples obtainedfrom the lysates of infected cells were subjected to nucleotidesequencing of the HIV genome.

EXAMPLE Determination of Nucleotide Sequences

Molecular cloning and determination of the nucleotide sequences of HIV-1strains passaged in the presence of anti-HIV-1 agents were performed asdescribed previously. Briefly, high-molecular-weight DNA was extractedfrom HIV-1-infected MT-4 cells by using the InstaGene Matrix (Bio-RadLaboratories, Hercules, Calif.) and was subjected to molecular cloning,followed by sequence determination. The first-round PCR mixtureconsisted of 1 of proviral DNA solution, 10 μl of Premix Taq (Ex Taqversion; Takara Bio, Inc., Otsu, Japan), and 10 pmol of each of thefirst PCR primers in a total volume of 20 ml. The PCR conditions usedwere an initial 5 cycles of 30 sec at 95° C., 2 min at 55° C., and 2 minat 72° C., and followed by 15 cycles of 30 sec at 95° C., 20 sec at 55°C., and 2 min at 72° C. The first-round PCR products were used directlyin the second round of PCR. The second-round PCR products were purifiedwith spin columns (MicroSpin S-400 HR columns; Amersham BiosciencesCorp., Piscataway, N.J.), cloned directly, and subjected to sequencingwith a cloned directly, and subjected to sequencing with a model 3130automated DNA sequencer (Applied Biosystems, Foster City, Calif.).

EXAMPLE Generation of FRET-Based HIV-1 Expression System.

The intermolecular fluorescence resonance energy transfer-basedHIV-1-expression assay employing cyan and yellow fluorescentprotein-tagged protease monomers (CFP and YFP, respectively) wasgenerated as described previously (Koh et al. 2007 J Biol Chem282:28709-20). Briefly, CFP- and YFP-tagged HIV-1 protease constructswere generated using BD Creatormi DNA Cloning Kits (BD Biosciences, SanJose, Calif.). For the generation of full 206 length molecularinfectious clones containing CFP- or YFP-tagged protease, the PCR207mediated recombination method was used (Fang et al. 1999 Nat Med5:239-42). A linker consisting of five alanines was inserted betweenprotease and fluorescent proteins. The phenylalanine-proline site thatHIV-1 protease cleaves was also introduced between the fluorescentprotein and RT. Thus obtained DNA fragments were subsequently joined byusing the PCR-mediated recombination reaction performed under thestandard condition for ExTaq polymerase (Takara Bio Inc., Otsu, Japan).The amplified PCR products were cloned into pCR-XL213 TOPO vectoraccording to the manufacturer's instructions (Gateway Cloning System;Invitrogen, Carlsbad, Calif.). PCR products were generated withpCR-XL-TOPO vector as templates, followed by digestion by both ApaI andSmaI, and the ApaI-SmaI fragment was introduced into pHIV-1_(NLSma)(Gatanaga et al. 2002 J Biol Chem 277:5952-61), generatingpHIV-PR_(WT CFP) and pHIV-PR_(WT YFP), respectively.

EXAMPLE FRET Procedure.

COS7 cells plated on EZ view cover-glass bottom culture plate (Iwaki,Tokyo) were transfected with pHIV-PR_(WT CFP) and pHIV-PR_(WT YFP) usingLipofectamine 2000 (Invitrogen, Carlsbad, Calif.) according tomanufacturer's instructions in the presence of various concentrations ofeach compound, cultured for 72 hrs, and analyzed under Fluoview FV500confocal laser scanning microscope (OLYMPUS Optical Corp, Tokyo) at roomtemperature as previously described. When the effect of each compoundwas analyzed by FRET, test compounds were added to the culture mediumsimultaneously with plasmid transfection. Results of FRET weredetermined by quenching of CFP (donor) fluorescence and an increase inYFP (acceptor) fluorescence (sensitized emission), since part of theenergy of CFP is transferred to YFP instead of being emitted. Thechanges in the CFP and YFP fluorescence intensity in the images ofselected regions were examined and quantified using Olympus FV500 Imagesoftware system (OLYMPUS Optical Corp). Ratios of intensities of CFPfluorescence after photobleaching to CFP fluorescence prior tophotobleaching (CFP_(A/B) ratios) were determined. When the CFP_(A/B)ratios were less than 1, it indicated that the association of the twosubunits did not occur and it was interpreted that protease dimerizationwas inhibited.

TABLE 6 EC₅₀ (μM) for Drug HIV-1_(LAI) HIV-2_(EHO) HIV-2_(ROD) CC₅₀ (μM)Selectivity Index APV 0.024 ± 0.008 0.12 ± 0.03 0.42 ± 0.10 >100 >4,170LPV 0.039 ± 0.006 0.035 ± 0.025 0.028 ± 0.004 >100 >2,560 TPV  0.17 ±0.005 0.30 ± 0.15 0.33 ± 0.07 54.1 ± 2.1 320 DRV  0.003 ± 0.0004 0.008 ±0.007 0.010 ± 0.004 >100 >33,300 GRL-216 0.002 ± 0.001 0.010 ± 0.0070.017 ± 0.001 48.8 ± 1.3 24,400 GRL-246 0.022 ± 0.005 N.D. N.D. 33.3 ±0.8 1,510 GRL-286 0.004 ± 0.001 0.018 ± 0.013 0.025 ± 0.002 33.1 ± 2.58,280 GRL-396 0.014 ± 0.001 N.D. N.D. 44.9 ± 4.1 3,200

MT-2 cells (2×10³) were exposed to 100 TCID₅₀s of HIV-1_(LAI),HIV-2_(EHO), or HIV-2_(ROD), and cultured in the presence of variousconcentrations of each PI, and EC₅₀s were determined by using the MTTassay. All assays were conducted in duplicate, and data shown representmean values (±1 standard deviation) derived from results of threeindependent experiments. Each selectivity index denotes a ratio of CC₅₀to EC₅₀ against HIV-1_(LAI) N.D., not done.

EXAMPLE GRL-216 and GRL-286 are potent against PI-selected laboratoryHIV-1 variants. HIV-1 variants that had been selected in vitro with eachof five FDA-approved PIs (SQV, NFV, APV, LPV and AZV) were obtained bypropagating a wild type laboratory HIV-1 strain, HIV-1NL4-3, in thepresence of increasing concentrations of each PI in MT-4 cells over37-60 passages in vitro. The thus obtained variants showed acquiredvarious PI resistance-associated amino acid substitutions in theprotease-encoding region of the viral genome. Each of the variants(HIV-1_(SQV5μM), HIV-1_(NFV5μM), HIV-1_(APV5μM), HIV-1_(LPV5μM), andHIV-1_(AZV5μM)) (Koh et al. 2009 Antimicrob Agents Chemother53:997-1006), was highly resistant to the corresponding PI, with whichthe variant was selected, with an EC₅₀ value of >1 μM; and the folddifferences of the EC₅₀ values, compared to the EC₅₀ value of each drugagainst HIV-1_(NL4-3), ranged from >22 to >250. The activities ofGRL-216 against all these variants except HIV-1_(APV5μM) were wellmaintained with fold-differences of 4 to 8. GRL-216 was virtually inertagainst HIV 1_(APV5μM), with an EC₅₀ value of >1 μM. GRL-286 was potentto HIV-1_(SQV5μM), HIV-1_(NFV5μM), and HIV-1_(AZV5μM), but the compoundwas less potent against HIV-1_(LPV5μM) and virtually inert againstHIV-1_(APV5μM).

EC₅₀ (μM) Virus SQV NFV APV LPV AZV HIV-1_(NL4-3) 0.005 ± 0.002 0.045 ±0.032 0.034 ± 0.001 0.046 ± 0.025  0.004 ± 0.0004 HIV-1_(SQV5 μM)  >1(>200)  0.51 ± 0.081 0.18 ± 0.10 0.12 ± 0.02  0.21 ± 0.065 (11)  (5) (2)(53)  HIV-1_(NPV5 μM) 0.003 ± 0.002  >1 (>22) 0.028 ± 0.005 0.037 ±0.004 0.027 ± 0.005 (1) (1) (1) (7) HIV-1_(APV5 μM) 0.026 ± 0.010 0.30 ±0.03  >1 (>29)  >1 (>22) 0.006 ± 0.002 (5) (7) (2) HIV-1_(LPV5 μM) 0.028± 0.007 0.32 ± 0.12 0.26 ± 0.07  >1 (>22) 0.036 ± 0.001 (6) (7) (8) (9)HIV-1_(AZV5 μM) 0.031 ± 0.005  >1 (>22) 0.28 ± 0.06  >1 (>22)  >1 (>250)(6) (8) Virus TPV DRV GRL-216 GRL-286 HIV-1_(NL4-3) 0.32 ± 0.07 0.003 ±0.0006  0.005 ± 0.001  0.009 ± 0.0006 HIV-1_(SQV5 μM) 0.022 ± 0.0090.003 ± 0.0002 0.020 ± 0.009 0.046 ± 0.021 (0.1) (1) (4) (5)HIV-1_(NPV5 μM) 0.039 ± 0.041 0.006 ± 0.0002 0.040 ± 0.017 0.041 ± 0.006(0.1) (2) (8) (5) HIV-1_(APV5 μM)  0.22 ± 0.089  0.29 ± 0.0018  >1(>200)  >1 (>111) (0.7) (97)  HIV-1_(LPV5 μM)  0.31 ± 0.030 0.025 ±0.0066 0.035 ± 0.005  0.35 ± 0.019 (1)   (8) (7) (39)  HIV-1_(AZV5 μM) 0.41 ± 0.047 0.009 ± 0.0025 0.023 ± 0.005 0.030 ± 0.006 (1)   (3) (5)(3)

Amino acid substitutions identified in the protease-encoding region ofHIV-1_(SQV-51M), HIV-1_(NFV-51M), HIV-1_(APV-51M), HIV-1_(LPV-51M) andHIV-1_(AZV-51M) compared to the consensus B sequence cited from the LosAlamos data base include L10I/G48V/I54V/A71V/I84V/L90M,L10F/D30N/K45I/A71V/T74S, L10F/M46I/I50V/A71V/I84V/L90M,L10F/M46I/I54V/V82A, andL23I/E34Q/K43I/M46I/I50L/G51A/L63P/A71V/V82A/T91A, respectively. MT-4cells (10⁴) were exposed to 100 TCID₅₀s of each HIV-1, and inhibition ofp24 Gag protein production by each drug was used as an end point.Numbers in parentheses represent n-fold changes in EC₅₀s for eachisolate compared to the EC₅₀s for wild-type HIV-1_(NL4-3). All assayswere conducted in duplicate or triplicate, and data shown represent meanvalues (±1 standard deviation) derived from results of three independentexperiments.

EXAMPLE GRL-216 and -286 exert potent activity against highlymulti-PI-resistant clinical HIV-1 strains. Previously isolated highlymultiple-PI-resistant clinical HIV-1 strains (HIV-1_(MDR)) includingHIV-1_(MDR/B), HIV-1_(MDR/C), HIV-1_(MDR/G), HIV-1_(MDR/TM),HIV-1_(MDR/MM), and HIV-1_(MDR/JSL), from patients with AIDS who hadfailed then-existing anti-HIV regimens after receiving 9 to 11anti-HIV-1 drugs over 32 to 83 months were evaluated. These clinicalstrains contained 9 to 14 amino acid substitutions in theprotease-encoding region, which have reportedly been associated withHIV-1 resistance to various PIs. The EC₅₀ values of IDV and LPV withthese multidrug-resistant clinical HIV-1 isolates were mostly >1 μM, andthe activity of other four PIs (SQV, APV, and AZV) was also found to besignificantly compromised, as examined in PHA-PBMCs as target cellsusing p24 production inhibition as an end point (Table 8). Both TPV andDRV had well maintained their activity and the fold-differences betweentheir EC₅₀ values against HIV-1_(ERS104pre) (wild-type) and thoseagainst multidrug-resistant clinical isolates ranged 1 to 9, while itwas noteworthy that the greatest EC₅₀ values of DRV was much lower(0.027 μM) than that of TPV (0.38 μM). GRL-216 and -286 exerted potentantiviral activity against HIV-1_(ERS104pre) with EC₅₀ values of as lowas 0.005 and 0.007 μM, respectively (Table 8). The potency of GRL-216and -286 against 5 PI-resistant variants (HIV-1_(MDR/B), HIV-1_(MDR/C),HIV-1_(MDR/G), HIV-1_(MDR/TM), and HIV-1_(MDR/MM)) was well maintainedwith the fold-differences of EC₅₀ values by 4 to 13, as compared totheir EC₅₀ values against HIV-1_(ERS104pre). However, both GRL-216 and-286 were less potent against HIV-1_(MDR/JSL) with the fold-differencesof EC₅₀ values by 15- and 30-fold, respectively. It was noted thatHIV-1_(MDR/JSL) was most resistant to all other PIs examined except TPVand DRV (Table 8).

TABLE 8 EC₅₀ (μM) Virus SQV IDV APV LPV AZV HIV-1

0.008 ± 0.005 0.043 ± 0.004 0.30 ± 0.005 0.034 ± 0.002 0.002 ± 0.001(wild-type SI) HIV-1

 (SI)  0.27 ± 0.073  >1 (>23)  >1 (>33)  >1 (>29) 0.20 ± 0.10 (34)(100)  HIV-1_(MDR/C) (SI) 0.032 ± 0.002  >1 (>23) 0.37 ± 0.011  >1 (>29)0.065 ± 0.008 (11) (12) (33) HIV-1_(MDR/G) (SI) 0.030 ± 0.002 0.34 ±0.14 0.43 ± 0.004 0.26 ± 0.04 0.033 ± 0.024  (4) (5) (14) (8) (17)HIV-1_(MDR/TM) 0.26 ± 0.04  >1 (>23) 0.32 ± 0.007  >1 (>29) 0.065 ±0.008 (SI) (33) (11) (33) HIV-1_(MDR/MM) 0.19 ± 0.05  >1 (>23) 0.21 ±0.222  >1 (>29)  0.18 ± 0.021 (NSI) (24)  (7) (39) HIV-1_(MDR/ )

0.30 ± 0.02   >1 (>23) 0.62 ± 0.02   >1 (>29)  0.43 ± 0.036 (NSI) (37)(21) (215)  Virus TPV DRV GRL-216 GRL-286 HIV-1

0.12 ± 0.03  0.003 ± 0.0002 0.005 ± 0.003 0.007 ± 0.002 (wild-type SI)HIV-1

 (SI)  0.18 ± 0.009 0.019 ± 0.012 0.037 ± 0.016 0.089 ± 0.016 (2) (6)(7) (13) HIV-1_(MDR/C) (SI)  0.38 ± 0.079 0.008 ± 0.006 0.044 ± 0.0020.029 ± 0.001 (3) (3) (9)  (4) HIV-1_(MDR/G) (SI) 0.24 ± 0.08 0.023 ±0.006 0.057 ± 0.012 0.028 ±0.004 (2) (5) (11)   (4) HIV-1_(MDR/TM) 0.38± 0.05 0.004 ± 0.001 0.027 ± 0.001 0.072 ± 0.014 (SI) (3) (1) (6) (10)HIV-1_(MDR/MM) 0.36 ± 0.06 0.011 ± 0.002 0.033 ± 0.010 0.055 ± 0.025(NSI) (3) (4) (7)  (8) HIV-1_(MDR/)

 0.23 ± 0.049 0.027 ± 0.011 0.073 ± 0.07   0.21 ± 0.032 (NSI) (2) (9)(15)  (30)

indicates data missing or illegible when filed

Amino acid substitutions identified in the protease-encoding regioncompared to the consensus type B sequence cited from the Los Alamosdatabase include L63P in HIV-1_(ERS104pre); L10I, K14R, L33I, M36I,M46I, F53I, K55R, I62V, L63P, A71V, G73S, V82A, L90M, and I93L inHIV-1_(MDR/B); L10I, I15V, K20R, L24I, M36I, M46L, I54V, I62V, L63P,K70Q, V82A, and L89M in HIV-1_(MDR/C); L10I, V11I, T12E, I15V, L19I,R41K, M46L, L63P, A71T, V82A, and L90M in HIV-1_(MDR/G); L10I, K14R,R41K, M46L, I54V, L63P, A71V, V82A, L90M, I93L in HIV-1_(MDR/TM); L10I,K43T, M46L, I54V, L63P, A71V, V82A, L90M, and Q92K in HIV-1_(MDR/MM);L10I, L24I, I33F, E35D, M36I, N37S, M46L, I54V, R57K, I62V, L63P, A71V,G73S, and V82A in HIV-1_(MDR/JSL). HIV-1_(ERS104pre) served as a sourceof wild-type HIV-1. EC₅₀s were determined by using PHA-PBMs as targetcells, and inhibition of p24 Gag protein production by each drug wasused as an end point. Numbers in parentheses represent n-fold changes ofEC₅₀s for each isolate compared to EC₅₀s for wild-typeHIV-1_(ERS104pre). All assays were conducted in duplicate or triplicate,and data shown represent mean values (±1 standard deviation) derivedfrom results of three independent experiments.

EXAMPLE GRL-216 and GRL-286 block the dimerization of HIV-1 protease. Inthe FRET-based HIV-1 expression system, COS7 cells were transfected withpHIV-PR_(WT CFP) and pHIV-PR_(WT YFP) and exposed to variousconcentrations of either of the drugs and the CFP_(A/B) ratios weredetermined at the end of 72-hr culture. In the absence of drug, theCFP_(A/B) ratios were virtually all above 1.0 with average figures of1.29 and 1.13 for GRL-216 and GRL-286, respectively (FIG. 1), indicatingthat protease dimerization occurred in the system. However, when thetransfected COS7 cells were exposed to greater than 0.1 μM of GRL-216,the average CFP_(A/B) ratios were all less than 1.0, indicating thatGRL-216 effectively blocked HIV-1 protease dimerization (FIG. 1).GRL-286 also effectively blocked the dimerization at the sameconcentration range.

EXAMPLE In vitro selection of HIV-1 variants resistant to four mcPIs.HIV-1 variants resistant to GRL-216, GRL-246, GRL-286, GRL-396 and APV,were selected out by propagating HIV-1_(NL4-3) in MT-4 cells in thepresence of increasing concentrations of each PI as previously described(Amano et al. 2007 Antimicrob Agents Chemother 51:2143-55). As shown inFIG. 2, HIV-1 variants that replicated in the presence of 1 μM of APV,GRL-396, GRL-246 and GRL-286 emerged by passages 20, 33, 33, and 37,respectively; however, the virus exposed to GRL-216 continued to befairly susceptible to GRL-216 even after 50 passages. Beyondapproximately 50 passages with GRL-216 exposure, the virus replicatedhighly poorly, and virtually failed to replicate at >0.26 μM,demonstrating that the emergence of GRL-216-resistant HIV-1 variant issubstantially delayed compared to APV and other mcPIs examined here.Determination of nucleotide sequence of the protease-encoding regiondisclosed that the variants resistant to APV (5 μM: passage 20) hadacquired previously reported mutations such as L10I, V32I, M46I, andI84V. By passage 5 with GRL-216 exposure, the wild-type protease genesequence in the virus was seen. However, by passage 10 and beyond, thevirus was seen to contain V82I substitution. As the passage proceeded,more amino acid substitutions were acquired. By passage 30, the virushad acquired L10I and I84V substitutions. By passage 50 (0.16 μMGRL-216: HIV_(216-P50)), the virus had further acquired L24I and M46Isubstitutions. By passage 60, the virus had gained L63P substitutions aswell. HIV-1 exposed to GRL-246 had acquired L10F, M46I, and T91S in theprotease 362 encoding region by passage 33 (1 μM GRL-246). HIV-1 exposedto GRL-286, by passage 37 (1 μM), had acquired L10F, M46L, I50V, andA71V in the protease-encoding region. The HIV-1 selected against GRL-396had acquired, by passage 33 (1 μM GRL-396), L10F, M46I, Q61K, V82I andI84V in the protease-encoding region. We also examined whether the virusacquired mutations in the Gag region at several passages of GRL-216selection. It was found that by passage 10, the virus had acquired theE107D substitution. By passage 25 and beyond, V35I, R275K and the p1/p6cleavage site substitution, L449F, had emerged. By passage 50, V390Demerged and persisted (data not shown). We also determined the aminoacid sequences of the gag region of each of the selected HIV variantswith direct base-sequencing. Two amino acid substitutions were seen incommon: V35I substitution was identified in GRL-216-, -246-, and-286-selected variants (by 25, 10, and 20 passages, respectively) andL449F substitution (Doyon et al. 1996 J Virol 70:3763-9) in GRL-216- and-286-selected variants (by 25 and 20 passages, respectively). Other thanthese two amino acid substitutions, only sporadic substitutions wereidentified in the gag region of the four variants (data not shown).

EXAMPLE Susceptibility of selected HIV-1 variants to various PIs. Asshown in Table 9, HIV_(216-P50) was resistant to GRL-216, with a24-fold-greater EC₅₀ (0.094 μM) relative to the EC₅₀ of GRL-216 againstHIV-1_(NL4-3). HIV_(216-P50) was more resistant to other three mcPIsexamined with the EC₅₀ fold difference values against HIV₁₄₆₋₁₀₄,HIV_(286-1 μM), and HIV_(396-1 μM) of 75, >250, and 18, respectively.However, HIV_(216-P50) was still susceptible to DRV with 3-folddifference relative to that against HIV-1_(N14-3). Interestingly, allthe four mcPI-selected HIV-1 variants including HIV_(216-P50) wereparadoxically more susceptible to TPV by factors of 3.3 to 10 relativeto the susceptibility of HIV-1_(NL4-3) against TPV (Table 9), suggestingthat the combination of GRL-216 (and other three mcPIs as well) and TPVcould exert complementarily augmented antiviral activity againstmcPI-resistant HIV-1 variants.

TABLE 9 EC₅₀ (μM) Virus APV NFV LPV AZV TPV HIV-1_(NL4-3) 0.032 ± 0.0010.040 ± 0.007 0.035 ± 0.004 0.0032 ± 0.0001 0.29 ± 0.05 HIV-1_(216-p50)0.26 ± 0.03 0.13 ± 0.04 0.19 ± 0.06 0.014 ± 0.003  0.029 ± 0.0004 (8)(4) (5) (4) (0.1) HIV-1_(246-1 μM)  0.34 ± 0.015  0.29 ± 0.006 0.33 ±0.04 0.032 ± 0.003 0.095 ± 0.089 (11)  (7) (8) (10)  (0.3)HIV-1_(286-1 μM)  >1 (>31) 0.37 ± 0.03  >1 (>23) 0.021 ± 0.012 0.037 ±0.001 (9) (7) (0.1) HIV-1_(396-1 μM)  0.35 ±0.0002 0.22 ± 0.02 0.28 ±0.05 0.024 ± 0.002 0.042 ± 0.002 (11) (6) (6) (8) (0.1) Virus DRVGRL-216 GRL-246 GRL-286 GRL-396 HIV-1_(NL4-3) 0.003 ± 0.001 0.004 ±0.001 0.033 ± 0.003 0.006 ± 0.005 0.017 ± 0.005 HIV-1_(216-p50) 0.009 ±0.006 0.094 ± 0.075 0.26 ± 0.05 0.19 ± 0.06 0.24 ± 0.06  (3) (24)  (8)(32) (14) HIV-1_(246-1 μM) 0.029 ± 0.001 0.30 ± 0.01 0.42 ± 0.09 0.33 ±0.03  0.37 ± 0.013 (10) (75) (12) (55) (22) HIV-1_(286-1 μM) 0.20 ± 0.05 >1 (>250)  >1 (>30)  >1 (>167)  >1 (>59) (67) HIV-1_(396-1 μM) 0.016 ±0.001 0.073 ± 0.011  0.37 ± 0.022 0.25 ± 0.05 0.52 ± 0.07  (5) (18) (11)(42) (31)

Amino acid substitutions identified in the protease-encoding region ofHIV-1_(216-P50), HIV-1_(246-11M), HIV-1_(286-11M), and HIV-1_(396-11M),compared to the consensus B sequence cited from the Los Alamos databaseinclude L10I/L24I/M46I/V82I/I84V, L10F/M46I/T91S, L10F/M46L/I50V/A71V,and L10F/M46M or I/Q61Q or K/V82I/I84V, respectively. MT-4 cells (10⁴)were exposed to 100 TCID₅₀s of each HIV-1, and inhibition of p24 Gagprotein production by each drug was used as an end point. Numbers inparentheses represent n-fold changes in EC₅₀s for each isolate comparedto the EC₅₀s for wild-type HIV-1_(NL4-3). All assays were conducted induplicate or triplicate, and data shown represent mean values (±1standard deviation) derived from results of three independentexperiments.

EXAMPLE V82I/I84V substitutions prevent GRL-216 from blocking proteasedimerization and play a role in HIV-1 resistance to GRL-216. It has beendiscovered herein that the GRL-216- and -396-selected HIV-1 variants hadacquired various amino acid substitutions prompted us to examine whethersuch amino acid substitutions affected the protease dimerizationinhibition of GRL-216. A pair (one with CFP and the other with YFP) ofrecombinant clones containing one of the following mutations: L24I,V82I, I84V, V82I/I84V, and L10I/L24I/M46L/L63P/V82I/I84V in the settingof the FRET-based HIV-1 expression assay were prepared. With L24I, V82I,or I84V alone, the activity of 0.1 μM GRL-216 to block proteasedimerization was not affected; however, with either set of V82I/I84V orL10I/L24I/M46L/L63P/V82I/I84V substitutions, GRL-216 failed to block thedimerization (FIG. 3). Thus, V82I/I84V substitutions prevented GRL-216from blocking protease dimerization and played a role in the emergenceof HIV-1 resistance to GRL-216.

EXAMPLE Structural analysis of interaction of GRL-216 and darunavir.Counter-ions were deleted from the crystal structures of HIV-1 proteasecomplexed with GRL-216 (PDB ID 3I6O) (16) and darunavir (PDB ID: 2IEN)(13). Bond orders were properly assigned to inhibitor molecules.Hydrogen's were added to all the heavy atoms and their positions wereoptimized in OPLS2005 force field with constraints on heavy atompositions. A cutoff distance of 3.0 Å between a polar hydrogen and anoxygen or nitrogen atom was used to determine the presence of hydrogenbonds. The structures were analyzed using Maestro version 9.0(Schrödinger, LLC, New York, N.Y., 2009).

EXAMPLE Structural Analysis of Interactions with GRL-216 with HIV-1protease. The crystal structures of GRL-216 (PDB ID 3160), and DRV (PDBID 2IEN) (34) were analyzed to gain insight to the similarity anddifferences in their interactions with HIV-1 protease. Hydrogens wereadded to the coordinates obtained from the PDB, and minimized withconstraints on the heavy atoms in OPLS2005 force field using Maestro 9.0(Schrodinger, LLC, New York). The bis-THF component of GRL-216 formshydrogen bonding interaction with D29 and D30 of the protease. Hydrogenbond interactions with D25, D25′, and a water molecule-mediated hydrogenbond interactions with I50 and I50′ were also observed. These hydrogenbond interactions were also observed for DRV. The P2′ ligands of DRV andGRL-216 were different and both of them formed hydrogen bondinteractions with different backbone atoms of D30′. The oxygen atom inthe methoxybenzene of GRL-216 formed hydrogen bond with the backbone NHof D30′, while the aniline nitrogen of darunavir hydrogen bonds to theoxygen of the backbone carbonyl. While the polar interactions of thesemolecules were similar, there appear to be subtle difference in thenon-polar interaction due to the presence of the macrocyclic ring inGRL-216. The macrocyclic ring occupied more volume at the S1′-S2′binding cavity of the protease, and formed more van der Waalsinteractions with V82 and I84 than the corresponding isopropyl group ofthe structurally related bis-THF-containing DRV. Without being bound bytheory, it is believed herein that the binding of GRL-216 to V82 and V84along with the polar interactions described herein significantlycontribute to its activity against HIV-1 protease. In addition, thoughwithout being bound by theory, it is believed herein that V82I and I84Vsubstitutions may have emerged during the in vitro selection processwith particularity.

Without being bound by theory, it is believed that the macrocylicportion of the inhibitors may allow for effective repacking in responseto side-chain mutations. Without being bound by theory, it is alsobelieved that such macrocyclic designs provide improved binding tomutants having an increased size of the S2 hydrophobic pocket. The X-raystructure and modeling studies of PI A-77003 indicated that the V82Amutant results in decreased van der Waals interaction with the phenylrings in both S1 and S1′-subsites (Kempf, 1991; Baldwin, 1995). There isalso evidence of repacking of inhibitor side chain and enzyme atoms inthe S1-subsite exhibited by the compounds described herein. Withoutbeing bound by theory, it is believed that enzyme flexibility inaccommodating alternate packing, allows a flexible macrocycle betweenthe P1′-side chain and the P2′-sulfonamide ring to fill in the S1′ andS2′-subsites (Baldwin, 1995). In particular, 12-16-membered saturatedand unsaturated macrocycles are described that effectively fill in theS1′-S2′ hydrophobic pocket of the enzyme active site while retaining allmajor interactions of PIs 1 and 2 with the protein backbone. Hereindescribed are examples of such inhibitors that maintain potency againstboth wild-type and mutant strains. PIs that incorporate variousmacrocycles to effectively fill in the enzyme active site are described.

EXAMPLE Biological Evaluation: The inhibitory potencies of acyclic andcyclic inhibitors were measured by the assay protocol of Toth andMarshall (Toth, M. V.; Marshall, G. R. A simple, continuous fluorometricassay for HIV protease. Int. J. Pep. Protien Res. 1990, 36, 544-55).Compounds that showed potent enzyme inhibition K_(i) values were furtherevaluated in an antiviral assay. Biological results for the acycliccompounds 13a-h are shown in TABLE 1. Enzyme inhibitory and antiviralactivity of inhibitors 14a-h and 15a-g are shown in TABLE 2. Enzymeinhibitory activity of E and Z isomers of 14a-c are shown in TABLE 3.Enzyme inhibitory activity of 19a-c, 20a-c, and 21a-c are shown in TABLE4. In TABLES 1-5 the following scale is used for K_(i) values: (−), >100nM; (+), ≦100 nM; (++), ≦10 nM; and (+++), ≦1 nM. In TABLES 1-4 thefollowing scale is used for IC₅₀ values: (−), >1000 nM; (+), ≦1000;(++), ≦100; and (+++), ≦10.

TABLE 1

IC₅₀ Corresponding Compound m n K_(i) (nM) Ring Size 13a 4 4 + − 15 13b4 3 + − 14 13c 4 2 ++ 13 13d 4 1 ++ 12 13e 1 4 ++ ++ 12 13f 1 3 +++ ++11 13g 1 2 +++ 10 13h 1 1 +++  9

TABLE 2

Ring Size Compound K_(i) IC₅₀ 15

  14a +++ ++ 14

  14b +++ +++ 13

  14c +++ +++ 12

  14d +++ ++ 12

  14e +++ ++ 11

  14f +++ ++ 10

  14g +++  9

  14h ++ 15

  15a +++ ++ 14

  15b +++ ++ 13

  15c +++ ++ 12

  15d +++ ++ 11

  15f +++ ++ 10

  15g +++ +++  9

  15h ++ ++

TABLE 3

Ring Size Compound K_(i) IC₅₀ 15

  14a-E +++ ++ 14

  14b-E +++ +++ 13

  14c-E +++ +++ 15

  14a-Z +++ − 14

  14b-Z +++ +++ 13

  14c-Z +++ +++

TABLE 4

Compound K_(i) IC₅₀ 19a

+ − 22a

++ − 19b

++ ++ 22b

+++ + 19c

++ − 22c

+++ ++ 20a

+ − 21a

++ − 20b

++ ++ 21b

++ +++ 20c

+++ +++ 21c

+++ ++

TABLE 5 Inhib- itor Structure K_(i)  0649A

++  0659A

− 00610A

− 00810A

− 00910A

− 01010A

− 01110A

+++ 01210A

+++ 01310A

+ 01410A

− 02110A

+ 02210A

+++

1-40. (canceled)
 41. A compound of the formula

or a pharmaceutically acceptable salt thereof; wherein R² and R³ arehydrogen; R⁵ is arylalkyl, which is optionally substituted; X⁴ iscarbonyl, S(O) or SO₂; X⁵ is oxygen; L¹ and L² are independentlyalkylene, which is optionally substituted; W is (H)C═C(H); Q² is aryl,which is optionally substituted with one or more substituents; and Z isa bicyclic heterocycle, which is optionally substituted.
 42. Thecompound of claim 41 wherein L¹ is CH₂(CH₂)_(m)CH₂; L² isCH₂(CH₂)_(n)CH₂; m is from 0 to about 4; and n is from 0 to about
 4. 43.The compound of claim 41 wherein L¹ is CH₂CH₂CH₂CH₂.
 44. The compoundclaim 42 wherein m is 2; and n is
 0. 45. The compound of claim 41wherein X⁴ is SO₂.
 46. The compound of claim 41 wherein Z is a bicycleheterocycle comprising at least one oxygen.
 47. The compound of claim 41wherein Z has the formula,

which is optionally substituted, wherein * indicates the point ofattachment; t is 0 to 4; W¹ is optionally substituted alkylene; W²represents optionally substituted alkylene; and W³ is optionallysubstituted alkylene.
 48. The compound of claim 41 wherein Z has formula

where * indicates the point of attachment.
 49. The compound of claim 41wherein R⁵ is optionally substituted benzyl.
 50. The compound of claim41 wherein Q² is optionally substituted 1,2-phenylene.
 51. The compoundof claim 41 wherein X⁴ is SO₂ and Q² is 4-methoxy-1,2-phenylene.
 52. Thecompound of claim 41 which is a compound of the formula

wherein R is selected from the group consisting of


53. A pharmaceutical composition comprising the compound of claim 41 ina therapeutically effective amount for treating HIV infection, and oneor more of a carrier, diluent, excipient therefor, or a combinationthereof.
 54. A method for treating a patient in need of relief fromHIV/AIDS disease, the method comprising the step of administering to thepatient a therapeutically effective amount of the compound of claim 41.55. The compound of claim 41 having the formula:

or a pharmaceutically acceptable salt thereof.
 56. The compound of claim41 having the formula:

or a pharmaceutically acceptable salt thereof.